Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars

ABSTRACT

The invention relates to a process for the preparation of a fermentation product from lignocellulosic material, comprising the following steps:
         a) optionally, pretreatment of the lignocellulosic material,   b) optionally, washing of the optionally pretreated lignocellulosic material,   c) enzymatic hydrolysis of the optionally washed and/or optionally pretreated lignocellulosic material using an enzyme composition comprising at least two cellulases and whereby the enzyme composition at least comprises LPMO, and optionally purifying the hydrolysed lignocellulosic material,   d) fermentation of the hydrolysed lignocellulosic material to produce a fermentation product, and   e) optionally, recovery of a fermentation product,
           wherein the amounts of formed hydrolysed oxidation products at the end of the enzymatic hydrolysis by the oxidation by LPMO of the lignocellulosic material containing cellulose and/or cello-oligosaccharides is kept between 3 to 80 g/kg glucan present in the lignocellulosic material by adding a suitable amount of oxygen after the pre-treatment and before and/or during the enzymatic hydrolysis to the lignocellulosic material, preferably the formed hydrolysed oxidation product is gluconic acid, an aldonic acid and/or geminal diol, more preferably the hydrolysed oxidation product is gluconic acid.

FIELD OF THE INVENTION

The invention relates to a process for the enzymatic hydrolysis oflignocellulosic material and fermentation of sugars.

BACKGROUND OF THE INVENTION

Lignocellulosic plant material, herein also called feedstock, is arenewable source of energy in the form of sugars that can be convertedinto valuable products e.g. sugars or biofuel, such as bioethanol.During this process, (ligno- or hemi)cellulose present in the feedstock,such as wheat straw, corn stover, rice hulls, etc., is converted intoreducing sugars by (hemi)cellulolytic enzymes, which then are optionallyconverted into valuable products such as ethanol by microorganisms likeyeast, bacteria and fungi.

Since the (hemi)cellulose is crystalline and entrapped in a network oflignin the conversion into reducing sugars is in general slow andincomplete. Typically, enzymatic hydrolysis of untreated feedstockyields sugars <20% of theoretical quantity. By applying a chemical andthermo-physical pretreatment, the (hemi)cellulose is more accessible forthe (hemi)cellulolytic enzymes and thus conversions go faster and athigher yields.

A typical ethanol yield from glucose, derived from pretreated cornstover, is 40 gallons of ethanol per 1000 kg of dry corn stover (BadgerP., Ethanol from cellulose: a general review, Trends in new crops andnew uses, 2002, J. Janick and A. Whipkey (eds.) ASHS Press, Alexandria,Va.) or 0.3 g ethanol per g feedstock. The maximum yield of ethanol oncellulose base is approximately 90%.

Cellulolytic enzymes—most of them are produced by species likeTrichoderma, Humicola and Aspergillus—are commercially used to convertpretreated feedstock into a mash containing insoluble (hemi)cellulose,reducing sugars made thereof, and lignin. Thermostable cellulolyticenzymes derived from Rasamsonia, have been used for degradinglignocellulosic feedstock and these enzymes are known for theirthermostability, see WO 2007/091231. The produced mash is used in afermentation during which the reducing sugars are converted into yeastbiomass (cells), carbon dioxide and ethanol. The ethanol produced inthis way is called bioethanol.

The common production of sugars from pretreated lignocelullosicfeedstock, the hydrolysis also called liquefaction, presaccharificationor saccharification, typically takes place during a process lasting 6 to168 hours (Kumar S., Chem. Eng. Technol. 32 (2009) 517-526) underelevated temperatures of 45 to 50° C. and non-sterile conditions. Duringthis hydrolysis, the cellulose present is partly (typically 30 to 95%,dependable on enzyme activity and hydrolysis conditions) converted intoreducing sugars. In case of inhibition of enzymes by compounds presentin the pretreated feedstock and by released sugars and to minimizethermal inactivation, this period of elevated temperature is minimizedas much as possible.

The fermentation following the hydrolysis takes place in a separatepreferably anaerobic process step, either in the same or in a differentvessel, in which temperature is adjusted to 30 to 33° C. (mesophilicprocess) to accommodate growth and ethanol production by microbialbiomass, commonly yeasts. During this fermentation process, theremaining (hemi)cellulosic material is converted into reducing sugars bythe enzymes already present from the hydrolysis step, while microbialbiomass and ethanol are produced. The fermentation is finished once(hemi)cellulosic material is converted into fermentable sugars and allfermentable sugars are converted into ethanol, carbon dioxide andmicrobial cells. This may take up to 6 days. In general the overallprocess time of hydrolysis and fermentation may amount up to 13 days.

The so-obtained fermented mash consists of non-fermentable sugars,non-hydrolysable (hemi)cellulosic material, lignin, microbial cells(most common yeast cells), water, ethanol, dissolved carbon dioxide.During the successive steps, ethanol is distilled from the mash andfurther purified. The remaining solid suspension is dried and used as,for instance, burning fuel, fertilizer or cattle feed.

WO 2010/080407 suggests treating cellulosic material with a cellulasecomposition under anaerobic conditions. Removal or exclusion of reactiveoxygen species may improve the performance of cellulose-hydrolyzingenzyme systems. Hydrolysis of cellulosic material, e.g. lignocellulose,by an enzyme composition can be reduced by oxidative damage tocomponents of the enzyme composition and/or oxidation of the cellulosicmaterial by, for example, molecular oxygen.

WO 2009/046538 discloses a method for treating lignocellulosic feedstockplant materials to release fermentable sugars using an enzymatichydrolysis process for treating the materials performed under vacuum andproducing a sugar rich process stream comprising reduced amounts ofvolatile sugar/fermentation inhibiting compounds such as furfural andacetic acid. Apart from removing volatile inhibitory compounds, othercompounds and/or molecules that are also removed include nitrogen,oxygen, argon and carbon dioxide.

With each batch of feedstock, enzymes are added to maximize the yieldand rate of fermentable sugars released from the pretreatedlignocellulosic feedstock during the given process time. In general,costs for enzymes production, feedstock to ethanol yields andinvestments are major cost factors in the overall production costs(Kumar S., Chem. Eng. Technol. 32 (2009) 517-526). Thus far, cost ofenzyme usage reduction is achieved by applying enzyme products from asingle or from multiple microbial sources (WO 2008/008793) with broaderand/or higher (specific) hydrolytic activity which use aims at a lowerenzyme need, faster conversion rates and/or a higher conversion yieldsand thus at overall lower bioethanol production costs. This requireslarge investments in research and development of these enzyme products.In case of an enzyme product composed of enzymes from multiple microbialsources, large capital investments are needed for production of eachsingle enzyme compound.

It is therefore desirable to improve the above process involvinghydrolysis and fermentation.

SUMMARY OF THE INVENTION

An object of the invention is therefore to provide a process in whichthe hydrolysis step is conducted at improved conditions. Another objectof the invention is to provide a process involving hydrolysis having areduced process time. Further object of the invention is to provide aprocess, wherein the dosage of enzyme may be reduced and at the sametime output of useful hydrolysis product is maintained at the same levelor even increased. Another object is to provide a process involvinghydrolysis, wherein the process conditions of the hydrolysis areoptimized. A still further object of the invention is to provide aprocess involving hydrolysis, wherein the output of useful hydrolysisproduct is increased using the same enzyme dosage. One or more of theseobjects are attained according to the invention.

The present invention provides a process for the preparation of a sugarproduct from lignocellulosic material, comprising the following steps:

-   -   a) optionally, pretreatment of the lignocellulosic material,    -   b) optionally, washing of the optionally pretreated        lignocellulosic material,    -   c) enzymatic hydrolysis of the optionally washed and/or        optionally pretreated lignocellulosic material using an enzyme        composition comprising at least two cellulases and whereby the        enzyme composition at least comprises LPMO, and    -   d) optionally, recovery of a glucose-containing composition,        wherein the amount of formed gluconic acid at the end of the        enzymatic hydrolysis by the oxidation by LPMO of the        lignocellulosic material containing cellulose and/or        cello-oligosaccharides is kept between 3 to 80 g/kg glucan        present in the lignocellulosic material by adding a suitable        amount of oxygen after the pretreatment and before and/or during        the enzymatic hydrolysis to the lignocellulosic material.

Furthermore the present invention provides a process for the preparationof a fermentation product from lignocellulosic material, comprising thefollowing steps:

-   -   a) optionally, pretreatment of the lignocellulosic material,    -   b) optionally, washing of the optionally pretreated        lignocellulosic material,    -   c) enzymatic hydrolysis of the optionally washed and/or        optionally pretreated lignocellulosic material using an enzyme        composition comprising at least two cellulases and whereby the        enzyme composition at least comprises LPMO, and optionally        purifying the hydrolysed lignocellulosic material,    -   d) fermentation of the hydrolysed lignocellulosic material to        produce a fermentation product, and    -   e) optionally, recovery of a fermentation product,        wherein the amount of formed gluconic acid at the end of the        enzymatic hydrolysis by the oxidation by LPMO of the        lignocellulosic material containing cellulose and/or        cello-oligosaccharides is kept between 3 to 80 g/kg glucan        present in the lignocellulosic material by adding a suitable        amount of oxygen after the pretreatment and before and/or during        the enzymatic hydrolysis to the lignocellulosic material.

Oxidation by LPMO of the lignocellulosic material results in oxidisedpolysaccharides which during the hydrolysis are hydrolysed into amongstothers glucose and oxidised glucose units such as gluconic acid or diol.In general, 1 molecule oxygen (O₂) gives one mol oxidation product.Oxygen can also be taken up by the feedstock (e.g. lignin).

Preferably, the oxygen is added during the enzymatic hydrolysis step c).

In a preferred embodiment the oxygen is added in the form of (gaseous)bubbles.

Surprisingly, according to the invention by the addition of oxygen it ispossible to attain many process advantages, including optimaltemperature conditions, reduced process time, reduced dosage of enzyme,re-use of enzymes, higher yields and other process optimizations,resulting in reduced costs.

In one embodiment of this process the fermentation time is 5 to 120hours. In an embodiment the stable enzyme composition used retainsactivity for 30 hours or more. According to a further embodiment thehydrolysis is preferably conducted at a temperature of 45° C. or more,more preferably at a temperature of 50° C. or more and still morepreferably at a temperature of 55° C. or more. In a preferred embodimentthe enzyme composition is derived from a fungus, preferably amicroorganism of the genus Rasamsonia or the enzyme compositioncomprises a fungal enzyme, preferably a Rasamsonia enzyme. The processof the invention will be illustrated in more detail below.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the present specification and the accompanying claims, thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows. The articles “a” and “an” are used herein to referto one or to more than one (i.e. to one or at least one) of thegrammatical object of the article. By way of example, “an element” maymean one element or more than one element.

In the context of the present invention “improved”, “increased”,“reduced” is used to indicate that the present invention shows anadvantage compared to the same situation, process or process conditionsexcept that no extra oxygen is added. Within the context of the presentinvention “measured under the same conditions” or “analysed under thesame conditions” etc. means that the process of the invention and thesame process without addition of oxygen are performed under the sameconditions (except the oxygen addition) and that the results of thepresent process, if compared to the process without oxygen addition, aremeasured using the same conditions, preferably by using the same assayand/or methodology, more preferably within the same or parallelexperiment. Conditions of the hydrolysis are an example of suchconditions.

In prior art it is suggested to improve the hydrolysis of cellulolyticmaterial by using anaerobic (WO 2010/080407) or vacuum (WO 2009/046538)conditions during the enzymatic hydrolysis. In the processes of bothdocuments the oxygen level was decreased. It has now surprisingly beenfound that the hydrolysis of the present invention shows results in animproved reaction product that gives higher amounts of (reduced) sugarproducts and/or desired fermentation products in the fermentationfollowing the hydrolysis as compared to a process wherein no oxygen isadded. In general, an increase of the glucose conversion is observed of5 to 15 w/w %.

Oxygen can be added in several ways. For example, oxygen can be added asoxygen gas, oxygen-enriched gas such as oxygen-enriched air or air.Oxygen can be added continuously or discontinuously. By oxygen “isadded” is meant that oxygen is added to the liquid phase (comprising thelignocellulosic material) in the hydrolysis reactor and not that oxygenis present in the headspace in the reactor above the liquid phase,whereby the oxygen has to diffuse from the headspace to the liquidphase. Preferably, oxygen is added or generated in the liquid phase(comprising the lignocellulosic material) in the hydrolysis reactor. Sopreferably, the oxygen is added as bubbles, most preferably as smallbubbles. In an embodiment the bubbles have a diameter of at least 0.5mm, at least 1 mm, at least 1.5 mm, at least 2 mm, at least 2.5 mm, atleast 3 mm, at least 3.5 mm, at least 4 mm, at least 4.5 mm, at least 5mm. In an embodiment the bubbles have a diameter of between 0.5 mm and500 mm, preferably between 0.5 mm and 400 mm, between 0.5 mm and 300 mm,between 0.5 mm and 200 mm, between 0.5 mm and 100 mm.

The inventors pose the hypothesis that in the (enzymatic) hydrolysis(step) amorphous and crystalline polysaccharides or cellulose arehydrolysed to sugars such as glucose. Amorphous polysaccharides are forexample converted to oligosaccharides by endogluconases, whereafter theoligosaccharides can be converted by cellobiohydrolase (CBH) andbeta-glucosidase (BG) to glucose. The conversion of the crystallinepolysaccharides may occur in parallel or sequential and continue evenwhen most of the amorphous polysaccharides are hydrolysed. According tothe present hypothesis especially the addition of oxygen in combinationwith LPMO is beneficial during the hydrolysis of the crystallinepolysaccharides for example in the degradation of the polysaccharidesinto oligosaccharides. Therefore, the addition of oxygen is very usefulespecially in the phase wherein crystalline polysaccharides areconverted by enzymes. Outside this phase, no addition of oxygen oradding less oxygen may be more efficient. This hypothesis is only givenas possible explanation of the effect noticed by the inventors and thepresent invention does not fall or stand with the correctness of thistheory.

The crystalline glucan structure can be opened by a lytic polysaccharidemonooxygenase (LPMO). This type of enzyme opens up the structure byoxidizing the glycosidic bonds and making it accessible for the othercellulolytic enzymes for further hydrolyzing the oligosaccharides intoglucose.

Most known LPMO's form aldonic acids, i.e. products oxidized at the C1position of the terminal sugar at the cleavage site. This oxidizedglucose unit is released as gluconic acid during hydrolysis. Inaddition, oxidation of the C4 and C6 of the non-reducing glucose unit atthe cleavage site has been reported. For instance, T. Isaksen et. al.(vide supra) reported the oxidation of the C4 position, the non-reducingend moiety, resulting in a keto-sugar at the C4 position, which is inequilibrium with a C4 geminal diol in water solution. The presentinventors pose that hydrolysed oxidation products. like for examplegluconic acid, are a measure for the performance of the applied LPMO inlignocellulose hydrolysis.

Surprisingly, the present inventors have found that optimallignocellulose hydrolysis (more than 70% glucan conversion) goes hand inhand with an optimal formation of oxidation products mentioned abovelike for instance gluconic acid. It will be evident that optimallignocellulose hydrolysis can only be achieved when (crystalline)cellulose and cello-oligosaccharides are hydrolysed optimally. Thisoptimal hydrolysis by the action of a LPMO will result in the formationof oxidation product, like gluconic acid. No oxidation product means aless efficient hydrolysis of (crystalline) glucan. However, too highlevels of oxidation products like gluconic acid will be at the expenseof glucose and therefore the glucose yield on (starting) glucan will godown. The amount of formed hydrolysed oxidation products by theoxidation of LPMO of cellulose and/or cello-oligosaccharides isadvantageously kept between 3 to 110 g/kg glucan present in thelignocellulosic material by adding a suitable amount of oxygen after thepretreatment and before and/or during the enzymatic hydrolysis to thelignocellulosic material. Preferably, the oxidation product is analdonic acid and/or geminal diol, more preferably the hydrolysedoxidation product is gluconic acid. Preferably, the amount of formedhydrolysed oxidation products by the oxidation of cellulose and/orcello-oligosaccharides is kept between 4 to 80 g/kg glucan present inthe lignocellulosic material, more preferably between 5 to 60 g/kgglucan present in the lignocellulosic material, even more preferablybetween 6 to 40 g/kg glucan present in the lignocellulosic material,still more preferably between 7 to 30 g/kg glucan present in thelignocellulosic material and most preferably between 8 to 25 g/kg glucanpresent in the lignocellulosic material. In another embodiment theamount of formed gluconic acid by the oxidation of cellulose and/orcello-oligosaccharides is kept between 3 to 110 g/kg glucan present inthe lignocellulosic material, preferably between 3 to 80 g/kg glucanpresent in the lignocellulosic material, preferably between 3 to 75 g/kgglucan present in the lignocellulosic material, preferably between 3 to70 g/kg glucan present in the lignocellulosic material, preferablybetween 3 to 65 g/kg glucan present in the lignocellulosic material,preferably between 3 to 60 g/kg glucan present in the lignocellulosicmaterial, preferably between 3 to 50 g/kg glucan present in thelignocellulosic material, preferably 4 to 50 g/kg glucan present in thelignocellulosic material, more preferably 4 to 30 g/kg glucan present inthe lignocellulosic material, even more preferably 4 to 20 g/kg glucanpresent in the lignocellulosic material and most preferably 5 to 10 g/kgglucan present in the lignocellulosic material.

By the process according to the present invention advantageously higheryields of glucose are obtained. Addition of higher amounts of oxygenwill result in more gluconic acid produced instead of glucose and on theother hand in case of lower amounts of oxygen the LPMO is not able tofunction optimally. Moreover, it was noticed that too high amounts ofoxidation products like gluconic acid may inhibit cellulases orhemicellulases or in case the hydrolysate is subsequently fermented, thegluconic acid may have a negative effect on the fermentation byinhibiting the microorganism, such as a yeast, used in the fermentation.The above amounts are the amounts at the end of hydrolysis

In general, the amount of oxygen added after the pretreatment and beforeand/or during the enzymatic hydrolysis to the lignocellulosic materialcan be controlled or varied in several ways. Restriction of the oxygensupplied is possible by adding only oxygen during part of the hydrolysistime. Another option is adding oxygen at a low concentration, forexample by using an mixture of air and recycled air (air leaving thehydrolysis reactor) or by “diluting” air with an inert gas. Increasingthe amount of oxygen added, can be obtained by addition of oxygen duringlonger periods of hydrolysis time, by adding the oxygen at a higherconcentration or by adding more air. Another option for changing theoxygen uptake is varying the hydrolysis temperature, a highertemperature will cause a lower maximal saturation concentration of theoxygen in the reactor content. Another way to manage the oxygenconcentration is to add an oxygen consumer or an oxygen generator. Incase the enzyme may be damaged by the presence or addition of oxygen,milder oxygen supply may be used. In that case, a balance can be foundbetween the improved glucose production and the enzyme performance. Theaddition of the oxygen to the cellulolytic material can be done beforeand/or during the enzymatic hydrolysis. In case oxygen is added ingaseous form, oxygen-containing gas can be introduced, for exampleblown, into the liquid hydrolysis reactor contents of cellulolyticmaterial. In another embodiment of invention the oxygen-containing gasis introduced into the liquid cellulolytic material stream that willenter the hydrolysis reactor. In still another embodiment of theinvention the oxygen containing gas is introduced together with thecellulolytic material that enters the hydrolysis reactor or with part ofthe liquid reactor contents that passes an external loop of the reactor.In most cases the addition of oxygen before entering the hydrolysisreactor is not sufficient enough and oxygen addition may be done duringthe hydrolysis as well. In another embodiment of the invention thegaseous phase present in the upper part of the reactor (head space) iscontinuously or discontinuously refreshed with the oxygen-containinggas. In the latter case (vigorous) mixing or stirring is needed to getthe oxygen as bubbles and/or by diffusion into the liquid reactorcontents preferably in combination with overpressure in the reactor. Ingeneral flushing the headspace with air in combination with (vigorous)mixing or stirring may introduce sufficient oxygen into the cellulosicmaterial in the hydrolysis reactor for reactors up to a size of 100liter to 1 m³. At larger scale, for example in a reactor of 50 m³ ormore, for example 100 m³, so much energy is needed for vigorous stirringthat from an economic point of view this will not be applied in acommercially operating process.

As described herein, the amount of formed gluconic acid at the end ofthe enzymatic hydrolysis by the oxidation by LPMO of the lignocellulosicmaterial containing cellulose and/or cello-oligosaccharides is keptbetween 3 to 80 g/kg glucan present in the lignocellulosic material byadding a suitable amount of oxygen after the pretreatment and beforeand/or during the enzymatic hydrolysis to the lignocellulosic material.In an embodiment “a suitable amount of oxygen” is 20-10,000 mmol oxygenper kg glucan, preferably 30-5,000 mmol oxygen per kg glucan, morepreferably 40-4,000 mmol oxygen per kg glucan and most preferably50-3,500 mmol oxygen per kg glucan. The amount of oxygen is the entireamount oxygen added during the enzymatic hydrolysis.

According to the present invention the oxygen may be added before thehydrolysis step, during part of the hydrolysis step, during the wholehydrolysis step or a combination of before or during the hydrolysisstep. Advantageously, the oxygen is added during the first half of thehydrolysis step. The addition of oxygen during only part of thehydrolysis may be done for example in case oxidation damage of theenzyme(s) occurs. In case the oxygen present in the hydrolysis reactorcontents or the sugar product or the hydrolysate formed in thehydrolysis step might influence or disturb the subsequent fermentationstep, oxygen addition may be done except for the last part of thehydrolysis and thus (most of) the oxygen is consumed before thehydrolysed biomass enters the fermentation reactor.

Several examples of aeration during the enzymatic hydrolysis process aregiven in the Examples to show the beneficial effect of the presentinvention. This beneficial effect is found for several substrates orfeedstocks and therefore believed to be present for the hydrolysis ofall kind of substrates or feedstocks.

Several examples of enzyme compositions for the enzymatic hydrolysisprocess are given in the Examples to show the beneficial effect of thepresent invention. This beneficial effect is found for several enzymecompositions and therefore believed to be present for all kind ofhydrolysing enzyme compositions.

To a further preferred embodiment of the invention the oxygenconcentration in the liquid phase (DO), wherein the lignocellulosicmaterial is present during the enzymatic hydrolysis, is at least 0.001mol/m³, preferably at least 0.002 mol/m³, more preferably at least 0.003mol/m³ and even more preferably more than 0.01 mol/m³, for example morethan 0.02 mol/m³ or 0.03 mol/m³. In reactors of less than 1 m³ DO valuesof below 0.01 mol/m³ or 0.02 mol/m³ will be obtained by slow stirring.Vigorous mixing or stirring at such scale introduces part of the gasphase of the headspace into the reaction liquid. For example, the mixingor stirring may create a whirlpool that draws oxygen into the liquid. Ingeneral, flushing the headspace with air in combination with (vigorous)mixing or stirring will introduce sufficient oxygen into the cellulosicmaterial in the hydrolysis reactor for reactors up to a size of 100liter to 1 m³. At larger scale, for example in a reactor of 50 m³ ormore, for example 100 m³, so much energy is needed for vigorous stirringthat from economic point of view this will not be applied in acommercially operating process. In general, in large reactors stirringor mixing without introducing air or oxygen will result in DO values ofless than 0.01 mol/m³.

To still another preferred embodiment of the invention during the oxygengeneration or production the oxygen concentration in the liquid phase(aeration or addition of oxygen), the oxygen concentration in the liquidphase wherein the lignocellulosic material is present during theenzymatic hydrolysis, is preferably at most 80% of the saturationconcentration of oxygen under the hydrolysis reaction conditions, morepreferably at most 0.12 mol/m³, still more preferably at most 0.09mol/m³, even more preferably at most 0.06 mol/m³, even still morepreferably at most 0.045 mol/m³ and most preferably at most 0.03 mol/m³.The above accounts for the situation when the oxygen transfer rate ofthe lignocellulosic material is larger than the oxygen uptake rate (OUR)of the lignocellulosic material. When the oxygen consumption(OUR) ishigher than the oxygen transfer rate, the oxygen concentration is 0mol/m³. Temperature and pressure will influence the DO. The preferredand exemplary mol/m³ values given above relate to normal atmosphericpressure and a temperature of about 62° C. The skilled person in the artwill appreciate favourable DO values on the basis of the presentteachings.

According to a further preferred embodiment of the invention oxygen isconsumed in an amount corresponding to between 20 and 5000 mmolmolecular oxygen per kg glucan present in the lignocellulosic material.Preferably, oxygen is consumed in an amount corresponding to between 22and 4500 mmol molecular oxygen per kg glucan present in thelignocellulosic material, between 24 and 4000 mmol molecular oxygen perkg glucan present in the lignocellulosic material, between 26 and 3500mmol molecular oxygen per kg glucan present in the lignocellulosicmaterial, between 28 and 3400 mmol molecular oxygen per kg glucanpresent in the lignocellulosic material. The oxygen is added after thepretreatment and before and/or during the enzymatic hydrolysis of thelignocellulosic material, preferably in an amount corresponding to atleast 30 mmol molecular oxygen per kg glucan present in thelignocellulosic material, more preferably in an amount corresponding toat least 40 mmol molecular oxygen per kg glucan present in thelignocellulosic material, and most preferably in an amount correspondingto at least 50 mmol molecular oxygen per kg glucan present in thelignocellulosic material. All oxygen that is added to the system will betransferred to the liquid and used for the hydrolysis. This amount canbe controlled by measuring and controlling the amount of air broughtinto the system.

According to a further preferred embodiment of the invention oxygen isconsumed in an amount corresponding to between 0.17 and 41.7 mmolmolecular oxygen per kg glucan present in the lignocellulosic materialper hour. Preferably, oxygen is consumed in an amount corresponding tobetween 0.18 and 37.5 mmol molecular oxygen per kg glucan present in thelignocellulosic material per hour, between 0.20 and 33.3 mmol molecularoxygen per kg glucan present in the lignocellulosic material per hour,between 0.22 and 29.2 mmol molecular oxygen per kg glucan present in thelignocellulosic material per hour, between 0.23 and 28.3 mmol molecularoxygen per kg glucan present in the lignocellulosic material per hour.More preferably, oxygen is consumed in an amount corresponding tobetween 0.36 and 27.8 mmol molecular oxygen per kg glucan present in thelignocellulosic material per hour. All oxygen that is added to thesystem will be transferred to the liquid and used for the hydrolysis.This amount can be controlled by measuring and controlling the amount ofair brought into the system. “Per hour” as used herein means per hour ofhydrolysis.

The oxygen addition in the form of air or other oxygen-containing gasaccording to the invention may also be used to at least partially stiror mix the hydrolysis reactor contents. Other ways of oxygen additioninclude the in situ oxygen generation. For example, the oxygen isgenerated by electrolysis, oxygen is produced enzymatically, preferablyby the addition of peroxide, or oxygen is produced chemically by forexample an oxygen generating system such as KHSO₅. For example, oxygenis produced from peroxide by catalase. The peroxide can be added in theform of dissolved peroxide or generated by an enzymatic or chemicalreaction. In case catalase is used as enzyme to produce oxygen, catalasepresent in the enzyme composition for the hydrolysis can be used orcatalase can be added for this purpose.

The present process of the invention shows advantages especially onpilot plant and industrial scale. According to an embodiment of theinvention the reactor for the enzymatic hydrolysis has a volume of 1 m³or more. Preferably, the reactor has a volume of at least 1 m³, at least2 m³, at least 3 m³, at least 4 m³, at least 5 m³, at least 6 m³, atleast 7 m³, at least 8 m³, at least 9 m³, at least 10 m³, at least 15m³, at least 20 m³, at least 25 m³, at least 30 m³, at least 35 m³, atleast 40 m³, at least 45 m³, at least 50 m³, at least 60 m³, at least 70m³, at least 75 m³, at least 80 m³, at least 90 m³, at least 100 m³, atleast 200 m³, at least 300 m³, at least 400 m³, at least 500 m³, atleast 600 m³, at least 700 m³, at least 800 m³, at least 900 m³, atleast 1000 m³, at least 1500 m³, at least 2000 m³, at least 2500 m³. Ingeneral, the reactor will be smaller than 3000 m³ or 5000 m³. Severalreactors may be used. The reactors used in the processes of the presentinvention may have the same volume, but also may have a differentvolume.

The inventor poses the theory that especially at large scaleinsufficient oxygen is available for the hydrolysis which might be dueto oxygen transfer limitations in the reactor, for example in thecellulolytic biomass. On lab-scale experiments this oxygen insufficiencymay play a less important role. The surface area (or oxygen contact areaof the reactor content) to reactor volume ratio is more favourable forsmall scale experiments than in large scale experiments. Moreover,mixing in small scale experiments is relatively easier than at largescale. During those small scale experiments also the transport of oxygenfrom the headspace of the hydrolysis reactor is faster than compared tothe situation in large scale experiments. This theory is only given aspossible explanation of the effect noticed by the inventors and thepresent invention does not fall or stands with the correctness of thistheory. According to a further embodiment of the invention the additionof oxygen may be used to control at least partially the hydrolysisprocess.

The process of the invention is advantageously applied in combinationwith the use of thermostable enzymes.

A “thermostable” enzyme means that the enzyme has a temperature optimum60° C. or higher, for example 70° C. or higher, such as 75° C. orhigher, for example 80° C. or higher such as 85° C. or higher. They mayfor example be isolated from thermophilic microorganisms, or may bedesigned by the skilled person and artificially synthesized. In oneembodiment the polynucleotides may be isolated or obtained fromthermophilic or thermotolerant filamentous fungi or isolated fromnon-thermophilic or non-thermotolerant fungi, but are found to bethermostable.

By “thermophilic fungus” is meant a fungus that grows at a temperatureof 50° C. or above. By “themotolerant” fungus is meant a fungus thatgrows at a temperature of 45° C. or above, having a maximum near 50° C.

Examples of thermophilic fungal strains are Rasamsonia emersonii(formerly known as Talaromyces emersoni). Talaromyces emersonii,Penicillium geosmithia emersonii and Rasamsonia emersonii are usedinterchangeably herein.

Suitable thermophilic or thermotolerant fungal cells may be a Humicola,Rhizomucor, Myceliophthora, Rasamsonia, Talaromyces, Thermomyces,Thermoascus or Thielavia cell, preferably a Rasamsonia emersonii cell.Preferred thermophilic or thermotolerant fungi are Humicola grisea var.thermoidea, Humicola lanuginosa, Myceliophthora thermophila, Papulasporathermophilia, Rasamsonia byssochlamydoides, Rasamsonia emersonii,Rasamsonia argillacea, Rasamsonia eburnean, Rasamsonia brevistipitata,Rasamsonia cylindrospora, Rhizomucor pusillus, Rhizomucor miehei,Talaromyces baciffisporus, Talaromyces leycettanus, Talaromycesthermophilus, Thermomyces lenuginosus, Thermoascus crustaceus,Thermoascus thermophilus Thermoascus aurantiacus and Thielaviaterrestris.

Thermophilic fungi are not restricted to a specific taxonomic order andoccur all over the fungal tree of life. Examples are Rhizomucor in theMucorales, Myceliophthora in Sordariales and Talaromyces, Thermomycesand Thermoascus in the Eurotiales (Mouchacca 1997). The majority ofTalaromyces species are mesophiles, but exceptions are species withinsections Emersonii and Thermophila. Section Emersonii includesTalaromyces emersonii, Talaromyces byssochlamydoides, Talaromycesbacillisporus and Talaromyces leycettanus, all of which grow well at 40°C. Talaromyces bacillisporus is thermotolerant, Talaromyces leycettanusis thermotolerant to thermophilic, and Talaromyces emersonii andTalaromyces byssochlamydoides are truly thermophilic (Stolk and Samson,1972). The sole member of Talaromyces section Thermophila, T.thermophilus, grows rapidly at 50° C. (Evans and Stolk, 1971; Evans,1971; Stolk and Samson, 1972). The current classification of thesethermophilic Talaromyces species is mainly based on phenotypic andphysiological characters, such as their ability to grow above 40° C.,ascospore color, the structure of ascornatal covering and the formationof a certain type of anamorph. Stolk and Samson (1972) stated that themembers of the section Emersonii have anamorphs of either Paecilomyces(T. byssochlamydoides and T. leycettanus) or Penicillium cylindrosporumseries (T. emersonii and T. bacillisporus). Later, Pitt (1979)transferred the species belonging to the Penicillium cylindrosporumseries to the genus Geosmithia, based on various characters such as theformation of conidia from terminal pores instead of on collula (necks),a character of Penicillium and Paecilomyces. Within the genusGeosmithia, only G. argillacea is thermotolerant, and Stolk et al.(1969) and Evans (1971) proposed a connection with members ofTalaromyces sect. Emersonii. The phylogenetic relationship of thethemophilic Talaromyces species within Talaromyces and theTrichocomaceae is unknown (see J. Houbraken, Antonie van Leeuwenhoek,2012 February; 101(2):403-21).

Rasamsonia is a new genus comprising thermotolerant and thermophilicTalaromyces and Geosmithia species (J. Houbraken et al., vida supra).Based on phenotypic, physiological and molecular data, Houbraken et al.proposed to transfer the species T. emersonii, T. byssochlamydoides, T.eburneus, G. argillacea and G. cylindrospora to Rasamsonia gen. nov.Talaromyces emersonii, Penicillium geosmithia emersonii and Rasamsoniaemersonii are used interchangeably herein.

Preferred thermophilic fungi are Rasamsonia byssochlamydoides,Rasamsonia emersonii, Thermomyces lenuginosus, Talaromyces thermophilus,Thermoascus crustaceus, Thermoascus thermophilus and Thermoascusaurantiacus.

“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK). The filamentous fungi arecharacterized by a mycelial wall composed of chitin, cellulose, glucan,chitosan, mannan, and other complex polysaccharides. Vegetative growthis by hyphal elongation and carbon catabolism is obligately aerobic.Filamentous fungal strains include, but are not limited to, strains ofAcremonium, Agaricus, Aspergillus, Aureobasidium, Beauvaria,Cephalosporium, Ceriporiopsis, Chaetomium paecilomyces, Chrysosporium,Claviceps, Cochiobolus, Coprinus, Cryptococcus, Cyathus, Emericella,Endothia, Endothia mucor, Filibasidium, Fusarium, Geosmithia,Gilocladium, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Panerochaete, Pleurotus, Podospora, Pyricularia, Rasamsonia, Rhizomucor,Rhizopus, Scylatidium, Schizophyllum, Stagonospora, Talaromyces,Thermoascus, Thermomyces, Thielavia, Tolypocladium, Trametes pleurotus,Trichoderma and Trichophyton.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). Examples of such strains includeAspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177,ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P.chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicilliumchrysogenum P2, Talaromyces emersonii CBS 393.64, Acremonium chrysogenumATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 orATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowense C1,Garg 27K, VKM F-3500-D, ATCC44006 and derivatives thereof.

An advantage of expression and production of the enzymes (for example atleast two, three or four different cellulases) in a suitablemicroorganism may be a high enzyme composition yield which can be usedin the process of the present invention.

According to the invention, by the addition of oxygen it is possible toattain many process advantages, including optimal temperatureconditions, reduced process time, reduced dosage of enzyme, re-use ofenzymes and other process optimizations, resulting in reduced costs.Advantageously the invention provides a process in which the hydrolysisstep is conducted at improved conditions. The invention also provides aprocess involving hydrolysis having a reduced process time. Furthermorethe invention provides a process, wherein the dosage of enzyme may bereduced and at the same time output of useful hydrolysis product ismaintained at the same level. Another advantage of the invention is thatthe present process involving hydrolysis may result in processconditions which are optimized. A still further advantage of theinvention is that the output of useful hydrolysis product of the processinvolving hydrolysis is increased using the same enzyme dosage.

Stable Enzyme Composition

Stable enzyme composition herein means that the enzyme compositionretains activity after 30 hours of hydrolysis reaction time, preferablyat least 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80% 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% of its initial activity after 30 hours ofhydrolysis reaction time. Preferably, the enzyme composition retainsactivity after 40, 50, 60, 70, 80, 90 100, 150, 200, 250, 300, 350, 400,450, 500 hours of hydrolysis reaction time.

In an embodiment the enzyme composition used in the processes of thepresent invention is derived from a fungus or the enzyme compositionused in the processes of the present invention comprises a fungalenzyme. In an embodiment the enzyme composition is derived from afilamentous fungus or the enzyme composition comprises a filamentousfungal enzyme. The processes of the invention are advantageously appliedin combination with enzyme compositions derived from a microorganism ofthe genus Rasamsonia, or the enzyme composition comprises a Rasamsoniaenzyme.

The enzyme composition may be prepared by fermentation of a suitablesubstrate with a suitable microorganism, e.g. Rasamsonia emersonii orAspergillus niger wherein the enzyme composition is produced by themicroorganism. The microorganism may be altered to improve or to makethe cellulase composition. For example, the microorganism may be mutatedby classical strain improvement procedures or by recombinant DNAtechniques. Therefore the microorganisms mentioned herein can be used assuch to produce the cellulase composition or may be altered to increasethe production or to produce an altered cellulase composition whichmight include heterologous cellulases, thus enzymes that are notoriginally produced by that microorganism. Preferably, a fungus, morepreferably a filamentous fungus, is used to produce the cellulasecomposition. Advantageously, a thermophilic or thermotolerantmicroorganism is used. Optionally, a substrate is used that induces theexpression of the enzymes in the enzyme composition during theproduction of the enzyme composition.

The enzyme composition is used to release sugars from lignocellulosethat comprises polysaccharides. The major polysaccharides are cellulose(glucans) and hemicelluloses (xylans, heteroxylans and xyloglucans). Inaddition, some hemicellulose may be present as glucomannans, for examplein wood-derived feedstocks. The enzymatic hydrolysis of thesepolysaccharides to soluble sugars, including both monomers andmultimers, for example glucose, cellobiose, xylose, arabinose,galactose, fructose, mannose, rhamnose, ribose, galacturonic acid,glucoronic acid and other hexoses and pentoses occurs under the actionof different enzymes acting in concert. By sugar product is meant theenzymatic hydrolysis product of the feedstock or lignocellulosicmaterial. The sugar product will comprise soluble sugars, including bothmonomers and multimers, preferably will comprise glucose. Examples ofother sugars are cellobiose, xylose, arabinose, galactose, fructose,mannose, rhamnose, ribose, galacturonic acid, glucoronic acid and otherhexoses and pentoses. The sugar product may be used as such or may befurther processed for example purified.

In addition, pectins and other pectic substances such as arabinans maymake up considerably proportion of the dry mass of typically cell wallsfrom non-woody plant tissues (about a quarter to half of dry mass may bepectins).

Cellulose is a linear polysaccharide composed of glucose residues linkedby β-1,4 bonds. The linear nature of the cellulose fibers, as well asthe stoichiometry of the β-linked glucose (relative to α) generatesstructures more prone to inter strand hydrogen bonding than the highlybranched α-linked structures of starch. Thus, cellulose polymers aregenerally less soluble, and form more tightly bound fibers than thefibers found in starch.

Enzymes that may be included in the stable enzyme composition used inthe invention are now described in more detail:

GH61, endoglucanases (EG) and exo-cellobiohydrolases (CBH) catalyze thehydrolysis of insoluble cellulose to products such ascellooligosaccharides (cellobiose as a main product), whilebeta-glucosidases (BG) convert the oligosaccharides, mainly cellobioseand cellotriose to glucose.

Hemicellulose is a complex polymer and its composition often varieswidely from organism to organism and from one tissue type to another. Ingeneral, a main component of hemicellulose is β-1,4-linked xylose, afive carbon sugar. However, this xylose is often branched at 0 to 3and/or 0 to 2 atom of xylose, and can be substituted with linkages toarabinose, galactose, mannose, glucuronic acid, galacturonic acid or byesterification to acetic acid (and esterification of ferulic acid toarabinose). Hemicellulose can also contain glucan, which is a generalterm for β-linked six carbon sugars (such as the β-(1,3)(1,4) glucansand heteroglucans mentioned previously) and additionally glucomannans(in which both glucose and mannose are present in the linear backbone,linked to each other by β-linkages).

Xylanases together with other accessory enzymes, for exampleα-L-arabinofuranosidases, feruloyl and acetylxylan esterases,glucuronidases, and β-xylosidases) catalyze the hydrolysis ofhemicelluloses.

Pectic substances include pectins, arabinans, galactans andarabinogalactans. Pectins are the most complex polysaccharides in theplant cell wall. They are built up around a core chain of α(1,4)-linkedD-galacturonic acid units interspersed to some degree with L-rhamnose.In any one cell wall there are a number of structural units that fitthis description and it has generally been considered that in a singlepectic molecule, the core chains of different structural units arecontinuous with one another.

The principal types of structural unit are: galacturonan(homogalacturonan) which may be substituted with methanol on thecarboxyl group and acetate on O-2 and O-3; rhamnogalacturonan I (RGI),in which galacturonic acid units alternate with rhamnose units carrying(1,4)-linked galactan and (1,5)-linked arabinan side-chains. Thearabinan side-chains may be attached directly to rhamnose or indirectlythrough the galactan chains; xylogalacturonan, with single xylosyl unitson O-3 of galacturonic acid (closely associated with RGI); andrhamnogalacturonan II (RGII), a particularly complex minor unitcontaining unusual sugars, for example apiose. An RGII unit may containtwo apiosyl residues which, under suitable ionic conditions, canreversibly form esters with borate.

A composition for use in a method of the invention comprises preferablyat least two activities, although typically a composition will comprisemore than two activities, for example, three, four, five, six, seven,eight, nine or more. Typically, a composition of the invention maycomprise at least two different celulases or two cellulases and at leastone hemicellulase. A composition of the invention may comprisecellulases, but no xylanases. In addition, a composition of theinvention may comprise auxiliary enzyme activity, i.e. additionalactivity which, either directly or indirectly leads to lignocellulosedegradation. Examples of such auxiliary activities are mentioned herein.

Thus, a composition for use in the invention may comprise GH61,endoglucanase activity and/or cellobiohydrolase activity and/orbeta-glucosidase activity. A composition for use in the invention maycomprise more than one enzyme activity in one or more of those classes.For example, a composition for use in the invention may comprise twoendoglucanase activities, for example, endo-1,3(1,4)β glucanase activityand endo-β-1,4-glucanase activity. Such a composition may also compriseone or more xylanase activities. Such a composition may comprise anauxiliary enzyme activity.

A composition for use in the current invention may be derived from afungus, such as a filamentous fungus such as Rasamsonia, such asRasamsonia emersonii. In an embodiment a core set of (lignocellulosedegrading) enzyme activities may be derived from Rasamsonia emersonii.Rasamsonia emersonii can provide a highly effective set of activities asdemonstrated herein for the hydrolysis of lignocellulosic material. Ifneeded, the set of activities can be supplemented with additional enzymeactivities from other sources. Such additional activities may be derivedfrom classical sources and/or produced by a genetically modifiedorganisms.

The activities in a composition for use in the invention may bethermostable. Herein, this means that the activity has a temperatureoptimum of about 60° C. or higher, for example about 70° C. or higher,such as about 75° C. or higher, for example about 80° C. or higher suchas 85° C. or higher. Activities in a composition for use in theinvention will typically not have the same temperature optima, butpreferably will, nevertheless, be thermostable.

In addition, enzyme activities in a composition for use in the inventionmay be able to work at low pH. For the purposes of this invention, lowpH indicates a pH of about 5.5 or lower, about 5 or lower, about 4.9 orlower, about 4.8 or lower, about 4.7 or lower, about 4.6 or lower, about4.5 or lower, about 4.4 or lower, about 4.3 or lower, about 4.2 orlower, about 4.1 or lower, about 4.0 or lower about 3.9 or lower, orabout 3.8 or lower, about 3.7 or lower, about 3.6 or lower, or about 3.5or lower.

Activities in a composition for use in the invention may be defined by acombination of any of the above temperature optima and pH values.

The composition used in a method of the invention may comprise, inaddition to the activities derived from Rasamsonia, a cellulase (forexample one derived from a source other than Rasamsonia) and/or ahemicellulase (for example one derived from a source other thanRasamsonia) and/or a pectinase.

The enzyme composition for use in the processes of the current inventionmay comprise a cellulase and/or a hemicellulase and/or a pectinase froma source other than Rasamsonia. They may be used together with one ormore Rasamsonia enzymes or they may be used without additionalRasamsonia enzymes being present.

For example, enzymes for use in the processes of the current inventionmay comprise a beta-glucosidase (BG) from Aspergillus, such asAspergillus oryzae, such as the one disclosed in WO 02/095014 or thefusion protein having beta-glucosidase activity disclosed in WO2008/057637, or Aspergillus fumigatus, such as the one disclosed as SEQID NO:2 in WO 2005/047499 or SEQ ID NO:5 in WO 2014/130812 or anAspergillus fumigatus beta-glucosidase variant, such as one disclosed inWO 2012/044915, such as one with the following substitutions: F100D,S283G, N456E, F512Y (using SEQ ID NO: 5 in WO 2014/130812 fornumbering), or Aspergillus aculeatus, Aspergillus niger or Aspergilluskawachi. In another embodiment the beta-glucosidase is derived fromPenicillium, such as Penicillium brasilianum disclosed as SEQ ID NO:2 inWO 2007/019442, or from Trichoderma, such as Trichoderma reesei, such asones described in U.S. Pat. No. 6,022,725, U.S. Pat. No. 6,982,159, U.S.Pat. No. 7,045,332, U.S. Pat. No. 7,005,289, US 2006/0258554 US2004/0102619. In an embodiment even a bacterial beta-glucosidase can beused. In another embodiment the beta-glucosidase is derived fromThielavia terrestris (WO 2011/035029) or Trichophaea saccata (WO2007/019442).

For example, enzymes for use in the processes of the current inventionmay comprise an endoglucanase (EG) from Trichoderma, such as Trichodermareesei; from Humicola, such as a strain of Humicola insolens; fromAspergillus, such as Aspergillus aculeatus or Aspergillus kawachii; fromErwinia, such as Erwinia carotovara; from Fusarium, such as Fusariumoxysporum; from Thielavia, such as Thielavia terrestris; from Humicola,such as Humicola grisea var. thermoidea or Humicola insolens; fromMelanocarpus, such as Melanocarpus albomyces; from Neurospora, such asNeurospora crassa; from Mycellophthora, such as Myceliophthorathermophila; from Cladorrhinum, such as Cladorrhinum foecundissimumand/or from Chrysosporium, such as a strain of Chrysosporiumlucknowense. In an embodiment even a bacterial endoglucanase can be usedincluding, but are not limited to, Acidothermus cellulolyficusendoglucanase (see WO 91/05039; WO 93/15186; U.S. Pat. No. 5,275,944; WO96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO 05/093050);Thermobifida fusca endoglucanase III (see WO 05/093050); andThermobifida fusca endoglucanase V (see WO 05/093050).

For example, enzymes for use in the processes of the current inventionmay comprise a cellobiohydrolase I from Aspergillus, such as Aspergillusfumigatus, such as the Cel7A CBH I disclosed in SEQ ID NO:6 in WO2011/057140 or SEQ ID NO:6 in WO 2014/130812, or from Trichoderma, suchas Trichoderma reesei.

For example, enzymes for use in the processes of the current inventionmay comprise a cellobiohydrolase II from Aspergillus, such asAspergillus fumigatus, such as the one in SEQ ID NO:7 in WO 2014/130812or from Trichoderma, such as Trichoderma reesei, or from Thielavia, suchas Thielavia terrestris, such as cellobiohydrolase II CEL6A fromThielavia terrestris.

For example, enzymes for use in the processes of the current inventionmay comprise a GH61 polypeptide (a lytic polysaccharide monooxygenase)from Thermoascus, such as Thermoascus aurantiacus, such as the onedescribed in WO 2005/074656 as SEQ ID NO:2 and SEQ ID NO:1 inWO2014/130812 and in WO 2010/065830; or from Thielavia, such asThielavia terrestris, such as the one described in WO 2005/074647 as SEQID NO: 8 or SEQ ID NO:4 in WO2014/130812 and in WO 2008/148131, and WO2011/035027; or from Aspergillus, such as Aspergillus fumigatus, such asthe one described in WO 2010/138754 as SEQ ID NO:2 or SEQ ID NO: 3 inWO2014/130812; or from Penicillium, such as Penicillium emersonii, suchas the one disclosed as SEQ ID NO:2 in WO 2011/041397 or SEQ ID NO:2 inWO2014/130812. Other suitable GH61 polypeptides include, but are notlimited to, Trichoderma reesei (see WO 2007/089290), Myceliophthorathermophila (see WO 2009/085935, WO 2009/085859, WO 2009/085864, WO2009/085868), Penicillium pinophilum (see WO 2011/005867), Thermoascussp. (see WO 2011/039319), and Thermoascus crustaceous (see WO2011/041504). In one aspect, the GH61 polypeptide is used in thepresence of a soluble activating divalent metal cation according to WO2008/151043, e.g. manganese sulfate. In one aspect, the GH61 polypeptideis used in the presence of a dioxy compound, a bicylic compound, aheterocyclic compound, a nitrogen-containing compound, a quinonecompound, a sulfur-containing compound, or a liquor obtained from apretreated cellulosic material such as pretreated corn stover.

Other cellulolytic enzymes that may be used in the processes of thepresent invention are described in WO 98/13465, WO 98/015619, WO98/015633, WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, WO2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S. Pat.No. 5,457,046, U.S. Pat. No. 5,648,263, and U.S. Pat. No. 5,686,593, toname just a few.

In addition, examples of xylanases useful in the processes of thepresent invention include, but are not limited to, xylanases fromAspergillus aculeatus (see WO 94/21785), Aspergillus fumigatus (see WO2006/078256), Penicillium pinophilum (see WO 2011/041405), Penicilliumsp. (see WO 2010/126772), Thielavia terrestris NRRL 8126 (see WO2009/079210), and Trichophaea saccata GH10 (see WO 2011/057083).Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa and Trichoderma reesei. Examples of acetylxylanesterases useful in the processes of the present invention include, butare not limited to, acetylxylan esterases from Aspergillus aculeatus(see WO 2010/108918), Chaetomium globosum, Chaetomium gracile, Humicolainsolens DSM 1800 (see WO 2009/073709), Hypocrea jecorina (see WO2005/001036), Myceliophtera thermophila (see WO 2010/014880), Neurosporacrassa, Phaeosphaeria nodorum and Thielavia terrestris NRRL 8126 (see WO2009/042846). Examples of feruloyl esterases (ferulic acid esterases)useful in the processes of the present invention include, but are notlimited to, feruloyl esterases form Humicola insolens DSM 1800 (see WO2009/076122), Neosartorya fischeri, Neurospora crassa, Penicilliumaurantiogriseum (see WO 2009/127729), and Thielavia terrestris (see WO2010/053838 and WO 2010/065448). Examples of arabinofuranosidases usefulin the processes of the present invention include, but are not limitedto, arabinofuranosidases from Aspergillus niger, Humicola insolens DSM1800 (see WO 2006/114094 and WO 2009/073383) and M. giganteus (see WO2006/114094). Examples of alpha-glucuronidases useful in the processesof the present invention include, but are not limited to,alpha-glucuronidases from Aspergillus clavatus, Aspergillus fumigatus,Aspergillus niger, Aspergillus terreus, Humicola insolens (see WO2010/014706), Penicillium aurantiogriseum (see WO 2009/068565) andTrichoderma reesei.

A composition for use in the invention may comprise one, two, three,four classes or more of cellulase, for example one, two three or four orall of a GH61, an endoglucanase (EG), one or two exo-cellobiohydrolase(CBH) and a beta-glucosidase (BG). A composition for use in theinvention may comprise two or more of any of these classes of cellulase.

A composition of the invention may comprise an activity which has adifferent type of cellulase activity and/or hemicellulase activityand/or pectinase activity than that provided by the composition for usein a method of the invention. For example, a composition of theinvention may comprise one type of cellulase and/or hemicellulaseactivity and/or pectinase activity provided by a composition asdescribed herein and a second type of cellulase and/or hemicellulaseactivity and/or pectinase activity provided by an additionalcellulose/hemicellulase/pectinase.

Herein, a cellulase is any polypeptide which is capable of degrading ormodifying cellulose. A polypeptide which is capable of degradingcellulose is one which is capable of catalysing the process of breakingdown cellulose into smaller units, either partially, for example intocellodextrins, or completely into glucose monomers. A cellulaseaccording to the invention may give rise to a mixed population ofcellodextrins and glucose monomers when contacted with the cellulase.Such degradation will typically take place by way of a hydrolysisreaction.

LPMO's (lytic polysaccharide monooxygenases) are recently classified byCAZy in family AA9 (Auxiliary Activity Family 9) or family AA10(Auxiliary Activity Family 10). As mentioned above LPMO is able to opena crystalline glucan structure. LPMO may also affectcello-oligosaccharides. PMO and LPMO are used herein interchangeably.GH61 (glycoside hydrolase family 61 or sometimes referred to EGIV)proteins are (lytic) oxygen-dependent polysaccharide monooxygenases(PMO's/LPMO's) according to the latest literature (Isaksen et al.,Journal of Biological Chemistry, vol. 289, no. 5, pp. 2632-2642). Oftenin literature, these proteins are mentioned to enhance the action ofcellulases on lignocellulose substrates. GH61 was originally classifiedas endoglucanase based on measurement of very weakendo-1,4-β-d-glucanase activity in one family member. The term “GH61” asused herein, is to be understood as a family of enzymes, which sharecommon conserved sequence portions and foldings to be classified infamily 61 of the well-established CAZy GH classification system(http://www.cazy.org/GH61.html). The glycoside hydrolase family 61 is amember of the family of glycoside hydrolases EC 3.2.1. GH61 are recentlynow reclassified by CAZy in family AA9 (Auxiliary Activity Family 9).GH61 is used herein as being part of the cellulases. CBM33 (family 33carbohydrate-binding module) is a LPMO (Isaksen et al., Journal ofBiological Chemistry, vol. 289, no. 5, pp. 2632-2642), CAZy has recentlyreclassified CBM33 in AA10 (Auxiliary Activity Family 10).

Herein, a hemicellulase is any polypeptide which is capable of degradingor modifying hemicellulose. That is to say, a hemicellulase may becapable of degrading or modifying one or more of xylan, glucuronoxylan,arabinoxylan, glucomannan and xyloglucan. A polypeptide which is capableof degrading a hemicellulose is one which is capable of catalysing theprocess of breaking down the hemicellulose into smaller polysaccharides,either partially, for example into oligosaccharides, or completely intosugar monomers, for example hexose or pentose sugar monomers. Ahemicellulase according to the invention may give rise to a mixedpopulation of oligosaccharides and sugar monomers when contacted withthe hemicellulase. Such degradation will typically take place by way ofa hydrolysis reaction.

Herein, a pectinase is any polypeptide which is capable of degrading ormodifying pectin. A polypeptide which is capable of degrading pectin isone which is capable of catalysing the process of breaking down pectininto smaller units, either partially, for example into oligosaccharides,or completely into sugar monomers. A pectinase according to theinvention may give rise to a mixed population of oligosacchardies andsugar monomers when contacted with the pectinase. Such degradation willtypically take place by way of a hydrolysis reaction.

Accordingly, a composition of the invention may comprise any cellulase,for example, a GH61, a cellobiohydrolase, an endo-β-1,4-glucanase, abeta-glucosidase or a β-(1,3)(1,4)-glucanase.

Herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide which iscapable of catalysing the hydrolysis of 1,4-β-D-glucosidic linkages incellulose or cellotetraose, releasing cellobiose from the ends of thechains. This enzyme may also be referred to as cellulase1,4-β-cellobiosidase, 1,4-β-cellobiohydrolase, 1,4-β-D-glucancellobiohydrolase, avicelase, exo-1,4-β-D-glucanase,exocellobiohydrolase or exoglucanase.

Herein, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptide which iscapable of catalysing the endohydrolysis of 1,4-β-D-glucosidic linkagesin cellulose, lichenin or cereal β-D-glucans. Such a polypeptide mayalso be capable of hydrolyzing 1,4-linkages in β-D-glucans alsocontaining 1,3-linkages. This enzyme may also be referred to ascellulase, avicelase, β-1,4-endoglucan hydrolase, β-1,4-glucanase,carboxymethyl cellulase, celludextrinase, endo-1,4-β-D-glucanase,endo-1,4-β-D-glucanohydrolase, endo-1,4-β-glucanase or endoglucanase.

Herein, a beta-glucosidase (EC 3.2.1.21) is any polypeptide which iscapable of catalysing the hydrolysis of terminal, non-reducingβ-D-glucose residues with release of β-D-glucose. Such a polypeptide mayhave a wide specificity for β-D-glucosides and may also hydrolyze one ormore of the following: a β-D-galactoside, an α-L-arabinoside, aβ-D-xyloside or a β-D-fucoside. This enzyme may also be referred to asamygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase.

Herein a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any polypeptide whichis capable of catalyzing the hydrolysis of 1,4-β-D-glucosidic linkagesin β-D-glucans containing 1,3- and 1,4-bonds. Such a polypeptide may acton lichenin and cereal β-D-glucans, but not on β-D-glucans containingonly 1,3- or 1,4-bonds. This enzyme may also be referred to aslicheninase, 1,3-1,4-β-D-glucan 4-glucanohydrolase, β-glucanase,endo-β-1,3-1,4 glucanase, lichenase or mixed linkage β-glucanase. Analternative for this type of enzyme is EC 3.2.1.6, which is described asendo-1,3(4)-beta-glucanase. This type of enzyme hydrolyses 1,3- or1,4-linkages in beta-D-glucanse when the glucose residue whose reducinggroup is involved in the linkage to be hydrolysed is itself substitutedat C-3. Alternative names include endo-1,3-beta-glucanase, laminarinase,1,3-(1,3;1,4)-beta-D-glucan 3 (4) glucanohydrolase; substrates includelaminarin, lichenin and cereal beta-D-glucans.

A composition of the invention may comprise any hemicellulase, forexample, an endoxylanase, a β-xylosidase, a α-L-arabionofuranosidase, anα-D-glucuronidase, an acetyl xylan esterase, a feruloyl esterase, acoumaroyl esterase, an α-galactosidase, a β-galactosidase, a β-mannanaseor a β-mannosidase.

Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capableof catalyzing the endohydrolysis of 1,4-β-D-xylosidic linkages inxylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative is EC 3.2.1.136, aglucuronoarabinoxylan endoxylanase, an enzyme that is able to hydrolyse1,4 xylosidic linkages in glucuronoarabinoxylans.

Herein, a β-xylosidase (EC 3.2.1.37) is any polypeptide which is capableof catalyzing the hydrolysis of 1,4-β-D-xylans, to remove successiveD-xylose residues from the non-reducing termini. Such enzymes may alsohydrolyze xylobiose. This enzyme may also be referred to as xylan1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-β-xylosidase orxylobiase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, an α-D-glucuronidase (EC 3.2.1.139) is any polypeptide which iscapable of catalyzing a reaction of the following form:alpha-D-glucuronoside+H(2)O=an alcohol+D-glucuronate. This enzyme mayalso be referred to as alpha-glucuronidase or alpha-glucosiduronase.These enzymes may also hydrolyse 4-O-methylated glucoronic acid, whichcan also be present as a substituent in xylans. Alternative is EC3.2.1.131: xylan alpha-1,2-glucuronosidase, which catalyses thehydrolysis of alpha-1,2-(4-O-methyl)glucuronosyl links.

Herein, an acetyl xylan esterase (EC 3.1.1.72) is any polypeptide whichis capable of catalyzing the deacetylation of xylans andxylo-oligosaccharides. Such a polypeptide may catalyze the hydrolysis ofacetyl groups from polymeric xylan, acetylated xylose, acetylatedglucose, alpha-napthyl acetate or β-nitrophenyl acetate but, typically,not from triacetylglycerol. Such a polypeptide typically does not act onacetylated mannan or pectin.

Herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:feruloyl-saccharide+H₂O=ferulate+saccharide. The saccharide may be, forexample, an oligosaccharide or a polysaccharide. It may typicallycatalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. β-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

Herein, a coumaroyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:coumaroyl-saccharide+H(2)O=coumarate+saccharide. The saccharide may be,for example, an oligosaccharide or a polysaccharide. This enzyme mayalso be referred to as trans-4-coumaroyl esterase, trans-β-coumaroylesterase, β-coumaroyl esterase or β-coumaric acid esterase. This enzymealso falls within EC 3.1.1.73 so may also be referred to as a feruloylesterase.

Herein, an α-galactosidase (EC 3.2.1.22) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

Herein, a β-galactosidase (EC 3.2.1.23) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingβ-D-galactose residues in β-D-galactosides. Such a polypeptide may alsobe capable of hydrolyzing α-L-arabinosides. This enzyme may also bereferred to as exo-(1->4)-β-D-galactanase or lactase.

Herein, a β-mannanase (EC 3.2.1.78) is any polypeptide which is capableof catalyzing the random hydrolysis of 1,4-β-D-mannosidic linkages inmannans, galactomannans and glucomannans. This enzyme may also bereferred to as mannan endo-1,4-β-mannosidase or endo-1,4-mannanase.

Herein, a β-mannosidase (EC 3.2.1.25) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingβ-D-mannose residues in β-D-mannosides. This enzyme may also be referredto as mannanase or mannase.

A composition of the invention may comprise any pectinase, for examplean endo-polygalacturonase, a pectin methyl esterase, anendo-galactanase, a beta-galactosidase, a pectin acetyl esterase, anendo-pectin lyase, pectate lyase, alpha-rhamnosidase, anexo-galacturonase, an expolygalacturonate lyase, a rhamnogalacturonanhydrolase, a rhamnogalacturonan lyase, a rhamnogalacturonan acetylesterase, a rhamnogalacturonan galacturonohydrolase, axylogalacturonase.

Herein, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide whichis capable of catalyzing the random hydrolysis of1,4-α-D-galactosiduronic linkages in pectate and other galacturonans.This enzyme may also be referred to as polygalacturonase pectindepolymerase, pectinase, endopolygalacturonase, pectolase, pectinhydrolase, pectin polygalacturonase, poly-α-1,4-galacturonideglycanohydrolase, endogalacturonase; endo-D-galacturonase orpoly(1,4-α-D-galacturonide) glycanohydrolase.

Herein, a pectin methyl esterase (EC 3.1.1.11) is any enzyme which iscapable of catalyzing the reaction: pectin+n H₂O=n methanol+pectate. Theenzyme may also been known as pectinesterase, pectin demethoxylase,pectin methoxylase, pectin methylesterase, pectase, pectinoesterase orpectin pectylhydrolase.

Herein, an endo-galactanase (EC 3.2.1.89) is any enzyme capable ofcatalyzing the endohydrolysis of 1,4-β-D-galactosidic linkages inarabinogalactans. The enzyme may also be known as arabinogalactanendo-1,4-β-galactosidase, endo-1,4-β-galactanase, galactanase,arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.

Herein, a pectin acetyl esterase is defined herein as any enzyme whichhas an acetyl esterase activity which catalyzes the deacetylation of theacetyl groups at the hydroxyl groups of GalUA residues of pectin

Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable ofcatalyzing the eliminative cleavage of (1→4)-α-D-galacturonan methylester to give oligosaccharides with4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducingends. The enzyme may also be known as pectin lyase, pectintrans-eliminase; endo-pectin lyase, polymethylgalacturonictranseliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGLor (1→4)-6-O-methyl-α-D-galacturonan lyase.

Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzingthe eliminative cleavage of (1→4)-α-D-galacturonan to giveoligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known polygalacturonictranseliminase, pectic acid transeliminase, polygalacturonate lyase,endopectin methyltranseliminase, pectate transeliminase,endogalacturonate transeliminase, pectic acid lyase, pectic lyase,α-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase,pectin trans-eliminase, polygalacturonic acid trans-eliminase or(1→4)-α-D-galacturonan lyase.

Herein, an alpha-rhamnosidase (EC 3.2.1.40) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingα-L-rhamnose residues in α-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T,α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.

Herein, exo-galacturonase (EC 3.2.1.82) is any polypeptide capable ofhydrolysis of pectic acid from the non-reducing end, releasingdigalacturonate. The enzyme may also be known asexo-poly-α-galacturonosidase, exopolygalacturonosidase orexopolygalacturanosidase.

Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable ofcatalyzing:(1,4-α-D-galacturonide)_(n)+H₂O=(1,4-α-D-galacturonide)_(n-1)+D-galacturonate.The enzyme may also be known as galacturan 1,4-α-galacturonidase,exopolygalacturonase, poly(galacturonate) hydrolase,exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase orpoly(1,4-α-D-galacturonide) galacturonohydrolase.

Herein, exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptidecapable of catalyzing eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate, i.e. de-esterified pectin. This enzyme may be known aspectate disaccharide-lyase, pectate exo-lyase, exopectic acidtranseliminase, exopectate lyase, exopolygalacturonicacid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-α-D-galacturonanreducing-end-disaccharide-lyase.

Herein, rhamnogalacturonan hydrolase is any polypeptide which is capableof hydrolyzing the linkage between galactosyluronic acid acid andrhamnopyranosyl in an endo-fashion in strictly alternatingrhamnogalacturonan structures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

Herein, rhamnogalacturonan lyase is any polypeptide which is anypolypeptide which is capable of cleaving α-L-Rhap-(1→4)-α-D-GalpAlinkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

Herein, rhamnogalacturonan acetyl esterase is any polypeptide whichcatalyzes the deacetylation of the backbone of alternating rhamnose andgalacturonic acid residues in rhamnogalacturonan.

Herein, rhamnogalacturonan galacturonohydrolase is any polypeptide whichis capable of hydrolyzing galacturonic acid from the non-reducing end ofstrictly alternating rhamnogalacturonan structures in an exo-fashion.

Herein, xylogalacturonase is any polypeptide which acts onxylogalacturonan by cleaving the β-xylose substituted galacturonic acidbackbone in an endo-manner. This enzyme may also be known asxylogalacturonan hydrolase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide which iscapable of catalyzing endohydrolysis of 1,5-α-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be known asendo-arabinase, arabinan endo-1,5-α-L-arabinosidase,endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase, endo-arabanase or1,5-α-L-arabinan 1,5-α-L-arabinanohydrolase.

A composition of the invention will typically comprise at least twocellulases and optionally at least one hemicellulase and optionally atleast one pectinase (one of which is a polypeptide according to theinvention). A composition of the invention may comprise a GH61, acellobiohydrolase, an endoglucanase and/or a beta-glucosidase. Such acomposition may also comprise one or more hemicellulases and/or one ormore pectinases.

In addition, one or more (for example two, three, four or all) of anamylase, a protease, a lipase, a ligninase, a hexosyltransferase, aglucuronidase or an expansin or a cellulose induced protein or acellulose integrating protein or like protein may be present in acomposition of the invention (these are referred to as auxiliaryactivities above).

“Protease” includes enzymes that hydrolyze peptide bonds (peptidases),as well as enzymes that hydrolyze bonds between peptides and othermoieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4, and are suitable for use in the inventionincorporated herein by reference. Some specific types of proteasesinclude, cysteine proteases including pepsin, papain and serineproteases including chymotrypsins, carboxypeptidases andmetalloendopeptidases.

“Lipase” includes enzymes that hydrolyze lipids, fatty acids, andacylglycerides, including phospoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

“Ligninase” includes enzymes that can hydrolyze or break down thestructure of lignin polymers. Enzymes that can break down lignin includelignin peroxidases, manganese peroxidases, laccases and feruloylesterases, and other enzymes described in the art known to depolymerizeor otherwise break lignin polymers. Also included are enzymes capable ofhydrolyzing bonds formed between hemicellulosic sugars (notablyarabinose) and lignin. Ligninases include but are not limited to thefollowing group of enzymes: lignin peroxidases (EC 1.11.1.14), manganeseperoxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and feruloylesterases (EC 3.1.1.73).

“Hexosyltransferase” (2.4.1-) includes enzymes which are capable ofcatalyzing a transferase reaction, but which can also catalyze ahydrolysis reaction, for example of cellulose and/or cellulosedegradation products. An example of a hexosyltransferase which may beused in the invention is a β-glucanosyltransferase. Such an enzyme maybe able to catalyze degradation of (1,3)(1,4)glucan and/or celluloseand/or a cellulose degradation product.

“Glucuronidase” includes enzymes that catalyze the hydrolysis of aglucoronoside, for example β-glucoronoside to yield an alcohol. Manyglucuronidases have been characterized and may be suitable for use inthe invention, for example β-glucuronidase (EC 3.2.1.31),hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamineglucuronidase (3.2.1.56), glycyrrhizinate β-glucuronidase (3.2.1.128) orα-D-glucuronidase (EC 3.2.1.139).

A composition for use in the invention may comprise an expansin orexpansin-like protein, such as a swollenin (see Salheimo et al., Eur. J.Biohem. 269, 4202-4211, 2002) or a swollenin-like protein.

Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Forthe purposes of this invention, an expansin-like protein orswollenin-like protein may comprise one or both of such domains and/ormay disrupt the structure of cell walls (such as disrupting cellulosestructure), optionally without producing detectable amounts of reducingsugars.

A composition for use in the invention may comprise a cellulose inducedprotein, for example the polypeptide product of the cip1 or cip2 gene orsimilar genes (see Foreman et al., J. Biol. Chem. 278(34), 31988-31997,2003), a cellulose/cellulosome integrating protein, for example thepolypeptide product of the cipA or cipC gene, or a scaffoldin or ascaffoldin-like protein. Scaffoldins and cellulose integrating proteinsare multi-functional integrating subunits which may organizecellulolytic subunits into a multi-enzyme complex. This is accomplishedby the interaction of two complementary classes of domain, i.e. acohesion domain on scaffoldin and a dockerin domain on each enzymaticunit. The scaffoldin subunit also bears a cellulose-binding module (CBM)that mediates attachment of the cellulosome to its substrate. Ascaffoldin or cellulose integrating protein for the purposes of thisinvention may comprise one or both of such domains.

A composition for use in the current invention may also comprise acatalase. The term “catalase” means a hydrogen-peroxide:hydrogen-peroxide oxidoreductase

(EC 1.11.1.6 or EC 1.11.1.21) that catalyzes the conversion of twohydrogen peroxides to oxygen and two waters. Catalase activity can bedetermined by monitoring the degradation of hydrogen peroxide at 240 nmbased on the following reaction: 2H₂O₂→2H₂O+O₂. The reaction isconducted in 50 mM phosphate pH 7.0 at 25° C. with 10.3 mM substrate(H₂O₂) and approximately 100 units of enzyme per ml. Absorbance ismonitored spectrophotometrically within 16-24 seconds, which shouldcorrespond to an absorbance reduction from 0.45 to 0.4. One catalaseactivity unit can be expressed as one micromole of H₂O₂ degraded perminute at pH 7.0 and 25° C.

A composition for use in a method of the invention may be composed of amember of each of the classes of enzymes mentioned above, severalmembers of one enzyme class, or any combination of these enzymes classesor helper proteins (i.e. those proteins mentioned herein which do nothave enzymatic activity per se, but do nevertheless assist inlignocellulosic degradation).

A composition for use in a method of the invention may be composed ofenzymes from (1) commercial suppliers; (2) cloned genes expressingenzymes; (3) complex broth (such as that resulting from growth of amicrobial strain in media, wherein the strains secrete proteins andenzymes into the media; (4) cell lysates of strains grown as in (3);and/or (5) plant material expressing enzymes. Different enzymes in acomposition of the invention may be obtained from different sources.

The enzymes can be produced either exogenously in microorganisms,yeasts, fungi, bacteria or plants, then isolated and added, for example,to lignocellulosic feedstock. Alternatively, the enzymes are produced,but not isolated, and crude cell mass fermentation broth, or plantmaterial (such as corn stover or wheat straw), and the like may be addedto, for example, the feedstock. Alternatively, the crude cell mass orenzyme production medium or plant material may be treated to preventfurther microbial growth (for example, by heating or addition ofantimicrobial agents), then added to, for example, a feedstock. Thesecrude enzyme mixtures may include the organism producing the enzyme.Alternatively, the enzyme may be produced in a fermentation that uses(pre-treated) feedstock (such as corn stover or wheat straw) to providenutrition to an organism that produces an enzyme(s). In this manner,plants that produce the enzymes may themselves serve as alignocellulosic feedstock and be added into lignocellulosic feedstock.

In the uses and methods described herein, the components of thecompositions described above may be provided concomitantly (i.e. as asingle composition per se) or separately or sequentially.

The invention thus relates to methods in which the composition describedabove are used and to uses of the composition in industrial processes.

In an embodiment of the processes according to the present invention theenzyme composition is in the form of a whole fermentation broth of afungus. In an embodiment the enzyme compositions may be a wholefermentation broth as described below. The whole fermentation broth canbe prepared from fermentation of non-recombinant and/or recombinantfilamentous fungi. In an embodiment the filamentous fungus is arecombinant filamentous fungus comprising one or more genes which can behomologous or heterologous to the filamentous fungus. In an embodiment,the filamentous fungus is a recombinant filamentous fungus comprisingone or more genes which can be homologous or heterologous to thefilamentous fungus wherein the one or more genes encode enzymes that candegrade a cellulosic substrate. The whole fermentation broth maycomprise any of the polypeptides or any combination thereof.

Preferably, the enzyme composition is whole fermentation broth whereinthe cells are killed. The whole fermentation broth may contain organicacid(s) (used for killing the cells), killed cells and/or cell debris,and culture medium.

Generally, the filamentous fungi is cultivated in a cell culture mediumsuitable for production of enzymes capable of hydrolyzing a cellulosicsubstrate. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable culture media, temperature rangesand other conditions suitable for growth and cellulase and/orhemicellulase and/or pectinase production are known in the art. Thewhole fermentation broth can be prepared by growing the filamentousfungi to stationary phase and maintaining the filamentous fungi underlimiting carbon conditions for a period of time sufficient to expressthe one or more cellulases and/or hemicellulases and/or pectinases. Onceenzymes, such as cellulases and/or hemicellulases and/or pectinases, aresecreted by the filamentous fungi into the fermentation medium, thewhole fermentation broth can be used. The whole fermentation broth ofthe present invention may comprise filamentous fungi. In someembodiments, the whole fermentation broth comprises the unfractionatedcontents of the fermentation materials derived at the end of thefermentation. Typically, the whole fermentation broth comprises thespent culture medium and cell debris present after the filamentous fungiis grown to saturation, incubated under carbon-limiting conditions toallow protein synthesis (particularly, expression of cellulases and/orhemicellulases and/or pectinases). In some embodiments, the wholefermentation broth comprises the spent cell culture medium,extracellular enzymes and filamentous fungi. In some embodiments, thefilamentous fungi present in whole fermentation broth can be lysed,permeabilized, or killed using methods known in the art to produce acell-killed whole fermentation broth. In an embodiment, the wholefermentation broth is a cell-killed whole fermentation broth, whereinthe whole fermentation broth containing the filamentous fungi cells arelysed or killed. In some embodiments, the cells are killed by lysing thefilamentous fungi by chemical and/or pH treatment to generate thecell-killed whole broth of a fermentation of the filamentous fungi. Insome embodiments, the cells are killed by lysing the filamentous fungiby chemical and/or pH treatment and adjusting the pH of the cell-killedfermentation mix to a suitable pH. In an embodiment, the wholefermentation broth comprises a first organic acid component comprisingat least one 1-5 carbon organic acid and/or a salt thereof and a secondorganic acid component comprising at least 6 or more carbon organic acidand/or a salt thereof. In an embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or any combination thereof and the second organic acid component isbenzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid,phenylacetic acid, a salt thereof, or any combination thereof.

The term “whole fermentation broth” as used herein refers to apreparation produced by cellular fermentation that undergoes no orminimal recovery and/or purification. For example, whole fermentationbroths are produced when microbial cultures are grown to saturation,incubated under carbon-limiting conditions to allow protein synthesis(e.g., expression of enzymes by host cells) and secretion into cellculture medium. Typically, the whole fermentation broth isunfractionated and comprises spent cell culture medium, extracellularenzymes, and microbial, preferably non-viable, cells.

If needed, the whole fermentation broth can be fractionated and the oneor more of the fractionated contents can be used. For instance, thekilled cells and/or cell debris can be removed from a whole fermentationbroth to provide a composition that is free of these components.

The whole fermentation broth may further comprise a preservative and/oranti-microbial agent. Such preservatives and/or agents are known in theart.

The whole fermentation broth as described herein is typically a liquid,but may contain insoluble components, such as killed cells, cell debris,culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedwhole fermentation broth.

In an embodiment, the whole fermentation broth may be supplemented withone or more enzyme activities that are not expressed endogenously, orexpressed at relatively low level by the filamentous fungi, to improvethe degradation of the cellulosic substrate, for example, to fermentablesugars such as glucose or xylose. The supplemental enzyme(s) can beadded as a supplement to the whole fermentation broth and the enzymesmay be a component of a separate whole fermentation broth, or may bepurified, or minimally recovered and/or purified.

In an embodiment, the whole fermentation broth comprises a wholefermentation broth of a fermentation of a recombinant filamentous fungioverexpressing one or more enzymes to improve the degradation of thecellulosic substrate. Alternatively, the whole fermentation broth cancomprise a mixture of a whole fermentation broth of a fermentation of anon-recombinant filamentous fungus and a recombinant filamentous fungusoverexpressing one or more enzymes to improve the degradation of thecellulosic substrate. In an embodiment, the whole fermentation brothcomprises a whole fermentation broth of a fermentation of a filamentousfungi overexpressing beta-glucosidase. Alternatively, the wholefermentation broth for use in the present methods and reactivecompositions can comprise a mixture of a whole fermentation broth of afermentation of a non-recombinant filamentous fungus and a wholefermentation broth of a fermentation of a recombinant filamentous fungioverexpressing a beta-glucosidase.

In an embodiment the process for the preparation of a sugar product fromlignocellulosic material comprises the following steps (a) optionally,pretreatment of the lignocellulosic material, (b) optionally, washing ofthe optionally pretreated lignocellulosic material, (c) producing anenzyme composition comprising at least two cellulases and whereby theenzyme composition at least comprises LPMO by culturing a fungus underconditions which allow for expression of the enzyme composition, (d)enzymatic hydrolysis of the optionally washed and/or optionallypretreated lignocellulosic material using the enzyme composition, and(e) optionally, recovery of a glucose-containing composition, whereinthe amounts of formed hydrolysed oxidation products at the end of theenzymatic hydrolysis by the oxidation by LPMO of the lignocellulosicmaterial containing cellulose and/or cello-oligosaccharides is keptbetween 3 to 110 g/kg glucan present in the lignocellulosic material byadding a suitable amount of oxygen after the pretreatment and beforeand/or during the enzymatic hydrolysis to the lignocellulosic material,preferably the formed hydrolysed oxidation product is gluconic acid, analdonic acid and/or geminal diol, more preferably the hydrolysedoxidation product is gluconic acid.

In an embodiment the process for the preparation of a fermentationproduct from lignocellulosic material comprises the following steps (a)optionally, pretreatment of the lignocellulosic material, (b)optionally, washing of the optionally pretreated lignocellulosicmaterial, (c) producing an enzyme composition comprising at least twocellulases and whereby the enzyme composition at least comprises LPMO byculturing a fungus under conditions which allow for expression of theenzyme composition, (d) enzymatic hydrolysis of the optionally washedand/or optionally pretreated lignocellulosic material using the enzymecomposition, (e) fermentation of the hydrolysed lignocellulosic materialto produce a fermentation product, and (f) optionally, recovery of afermentation product, wherein the amounts of formed hydrolysed oxidationproducts at the end of the enzymatic hydrolysis by the oxidation by LPMOof the lignocellulosic material containing cellulose and/orcello-oligosaccharides is kept between 3 to 110 g/kg glucan present inthe lignocellulosic material by adding a suitable amount of oxygen afterthe pre-treatment and before and/or during the enzymatic hydrolysis tothe lignocellulosic material, preferably the formed hydrolysed oxidationproduct is gluconic acid, an aldonic acid and/or geminal diol, morepreferably the hydrolysed oxidation product is gluconic acid.

As indicated above, in a preferred embodiment the fungus is afilamentous fungus, preferably the fungus belongs to the genusRasamsonia or Aspergillus. In an embodiment the culturing of the fungusis conducted under aerobic conditions. A person skilled in the art iswell aware of fermentor designs for aerobic cultivation such as forinstance stirred tanks and bubble columns. Generally, the fungi arecultivated in a cell culture medium suitable for production of theenzyme composition of interest. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable culturemedia, temperature ranges and other conditions suitable for growth andenzyme production are known in the art. Examples thereof are describedherein. The enzyme composition can be prepared by growing the fungi tostationary phase and maintaining the fungi under limiting carbonconditions for a period of time sufficient to express the enzymes. Oncethe enzymes of interest are secreted by the fungi into the fermentationmedium, the enzyme composition can be used. The process step ofproducing an enzyme composition comprising at least two cellulases andwhereby the enzyme composition at least comprises LPMO by culturing afungus under conditions which allow for expression of the enzymecomposition as described herein can be preceded by a process forpropagating the fungus. Propagation may comprise several steps in shakeflasks, small containers and large containers.

Lignocellulosic Material

Lignocellulosic material herein includes any lignocellulosic and/orhemicellulosic material. Lignocellulosic material suitable for use asfeedstock in the invention includes biomass, e.g. virgin biomass and/ornon-virgin biomass such as agricultural biomass, commercial organics,construction and demolition debris, municipal solid waste, waste paperand yard waste. Common forms of biomass include trees, shrubs andgrasses, wheat, wheat straw, sugar cane, cane straw, sugar cane bagasse,sugar cane trash, switch grass, miscanthus, energy cane, corn, cornstover, corn husks, corn cobs, canola stems, soybean stems, sweetsorghum, corn kernel including fiber from kernels, products andby-products from milling of grains such as corn, wheat and barley(including wet milling and dry milling) often called “bran or fibre” aswell as municipal solid waste, waste paper and yard waste. The biomasscan also be, but is not limited to, herbaceous material, agriculturalresidues, forestry residues, municipal solid wastes, waste paper, andpulp and paper mill residues. “Agricultural biomass” includes branches,bushes, canes, corn and corn husks, energy crops, forests, fruits,flowers, grains, grasses, herbaceous crops, leaves, bark, needles, logs,roots, saplings, short rotation woody crops, shrubs, switch grasses,trees, vegetables, fruit peels, vines, sugar beet pulp, wheat midlings,oat hulls, and hard and soft woods (not including woods with deleteriousmaterials). In addition, agricultural biomass includes organic wastematerials generated from agricultural processes including farming andforestry activities, specifically including forestry wood waste.Agricultural biomass may be any of the aforementioned singularly or inany combination or mixture thereof.

Cellulose is an organic compound with the formula (C₆H₁₀O₅)_(n), apolysaccharide consisting of a linear chain of several hundred to overten thousand β(1→4) linked D-glucose units. A glucan molecule is apolysaccharide of D-glucose monomers linked by glycosidic bonds. Hereinglucan and cellulose are used interchangeably for a polysaccharide ofD-glucose monomers linked by glycosidic bonds. Methods for thequantitative analysis of glucan or polysaccharide compositions arewell-known and described in the art and are for example summarized inCarvalho de Souza et al., Carbohydrate Polymers 95 (2013) 657-663. Ingeneral, 50 to 70% of the glucan is crystalline cellulose, the remainderis amorphous cellulose.

Pretreatment

The lignocellulosic material used in the present invention may be washedand/or pretreated. The feedstock may optionally be pretreated with heat,mechanical and/or chemical modification or any combination of suchmethods in order to enhance the accessibility of the substrate toenzymatic hydrolysis and/or hydrolyse the hemicellulose and/orsolubilize the hemicellulose and/or cellulose and/or lignin, in any wayknown in the art. In one embodiment, the pretreatment is conductedtreating the lignocellulose with steam explosion, hot water treatment ortreatment with dilute acid or dilute base.

In an embodiment the lignocellulosic material is pretreated beforeand/or during the enzymatic hydrolysis. Pretreatment methods are knownin the art and include, but are not limited to, heat, mechanical,chemical modification, biological modification and any combinationthereof. Pretreatment is typically performed in order to enhance theaccessibility of the lignocellulosic material to enzymatic hydrolysisand/or hydrolyse the hemicellulose and/or solubilize the hemicelluloseand/or cellulose and/or lignin, in the lignocellulosic material. In anembodiment, the pretreatment comprises treating the lignocellulosicmaterial with steam explosion, hot water treatment or treatment withdilute acid or dilute base. Examples of pretreatment methods include,but are not limited to, steam treatment (e.g. treatment at 100-260° C.,at a pressure of 7-45 bar, at neutral pH, for 1-10 minutes), dilute acidtreatment (e.g. treatment with 0.1-5% H₂SO₄ and/or SO₂ and/or HNO₃and/or HCl, in presence or absence of steam, at 120-200° C., at apressure of 2-15 bar, at acidic pH, for 2-30 minutes), organosolvtreatment (e.g. treatment with 1-1.5% H₂SO₄ in presence of organicsolvent and steam, at 160-200° C., at a pressure of 7-30 bar, at acidicpH, for 30-60 minutes), lime treatment (e.g. treatment with 0.1-2%NaOH/Ca(OH)₂ in the presence of water/steam at 60-160° C., at a pressureof 1-10 bar, at alkaline pH, for 60-4800 minutes), ARP treatment (e.g.treatment with 5-15% NH₃, at 150-180° C., at a pressure of 9-17 bar, atalkaline pH, for 10-90 minutes), AFEX treatment (e.g. treatmentwith >15% NH₃, at 60-140° C., at a pressure of 8-20 bar, at alkaline pH,for 5-30 minutes).

Washing Step

Optionally, the process according to the invention comprises a washingstep. The optional washing step may be used to remove water solublecompounds that may act as inhibitors for the fermentation step. Thewashing step may be conducted in known manner.

The lignocellulosic material may be washed. In an embodiment thelignocellulosic material may be washed after the pretreatment. Thewashing step may be used to remove water soluble compounds that may actas inhibitors for the fermentation and/or hydrolysis step. The washingstep may be conducted in manner known to the skilled person. Next towashing, other detoxification methods do exist. The pretreatedlignocellulosic material may also be detoxified by any (or anycombination) of these methods which include, but are not limited to,solid/liquid separation, vacuum evaporation, extraction, adsorption,neutralization, overlimiting, addition of reducing agents, addition ofdetoxifying enzymes such as laccases or peroxidases, addition ofmicroorganisms capable of detoxification of hydrolysates.

Enzymatic Hydrolysis

The enzyme composition used in the process of the invention canextremely effectively hydrolyze lignocellulolytic material, for examplecorn stover, wheat straw, cane straw, and/or sugar cane bagasse, whichcan then be further converted into a useful product, such as ethanol,biogas, butanol, lactic acid, a plastic, an organic acid, a solvent, ananimal feed supplement, a pharmaceutical, a vitamin, an amino acid, anenzyme or a chemical feedstock. Additionally, intermediate products froma process following the hydrolysis, for example lactic acid asintermediate in biogas production, can be used as building block forother materials. The present invention is exemplified with theproduction of ethanol but this is done as exemplification only ratherthan as limitation, the other mentioned useful products can be producedequally well.

The process according to the invention comprises an enzymatic hydrolysisstep. The enzymatic hydrolysis includes, but is not limited to,hydrolysis for the purpose of liquefaction of the feedstock andhydrolysis for the purpose of releasing sugar from the feedstock orboth. In this step optionally pretreated and optionally washedlignocellulosic material is brought into contact with the enzymecomposition according to the invention. Depending on the lignocellulosicmaterial and the pretreatment, the different reaction conditions, e.g.temperature, enzyme dosage, hydrolysis reaction time and dry matterconcentration, may be adapted by the skilled person in order to achievea desired conversion of lignocellulose to sugar. Some indications aregiven hereafter.

In an embodiment the enzymatic hydrolysis comprises at least aliquefaction step wherein the lignocellulosic material is hydrolysed inat least a first container, and a saccharification step wherein theliquefied lignocellulosic material is hydrolysed in the at least firstcontainer and/or in at least a second container. Saccharification can bedone in the same container as the liquefaction (i.e. the at least firstcontainer), it can also be done in a separate container (i.e. the atleast second container). So, in the enzymatic hydrolysis of theprocesses according to the present invention liquefaction andsaccharification may be combined. Alternatively, the liquefaction andsaccharification may be separate steps. Liquefaction andsaccharification may be performed at different temperatures, but mayalso be performed at a single temperature. In an embodiment thetemperature of the liquefaction is higher than the temperature of thesaccharification. Liquefaction is preferably carried out at atemperature of 60-75° C. and saccharification is preferably carried outat a temperature of 50-65° C.

The enzymes used in the enzymatic hydrolysis may be added before and/orduring the enzymatic hydrolysis. In case the enzymatic hydrolysiscomprises a liquefaction step and saccharification step, additionalenzymes may be added during and/or after the liquefaction step. Theadditional enzymes may be added before and/or during thesaccharification step. Additional enzymes may also be added after thesaccharification step.

In one aspect of the invention the hydrolysis is conducted at atemperature of 45° C. or more, 50° C. or more, 55° C. or more, 60° C. ormore, 65° C. or more, or 70° C. or more. The high temperature duringhydrolysis has many advantages, which include working at the optimumtemperature of the enzyme composition, the reduction of risk of(bacterial) contamination, reduced viscosity, smaller amount of coolingwater required, use of cooling water with a higher temperature, re-useof the enzymes and more.

The viscosity of the lignocellulosic material in the one or morecontainers used for the enzymatic hydrolysis is kept between 10 and 4000cP, between 10 and 2000 cP, preferably between 10 and 1000 cP.

In case the process comprises an enzymatic hydrolysis comprising aliquefaction step and a saccharification step, the viscosity of thelignocellulosic material in the liquefaction step is kept between 10 and4000 cP, between 10 and 2000 cP, preferably between 10 and 1000 cPand/or the viscosity of the lignocellulosic material in thesaccharification step is kept between 10 and 1000 cP, between 10 and 900cP, preferably between 10 and 800 cP.

The viscosity can be determined with a Brookfield DV III Rheometer atthe temperature used for the hydrolysis.

In a further aspect of the invention, the amount of enzyme compositionadded (herein also called enzyme dosage or enzyme load) is low. In anembodiment the amount of enzyme is 6 mg protein/g dry matter weight orlower, 5 mg protein/g dry matter or lower, 4 mg protein/g dry matter orlower, 3 mg protein/g dry matter or lower, 2 mg protein/g dry matter orlower, or 1 mg protein/g dry matter or lower (expressed as protein in mgprotein/g dry matter). In an embodiment, the amount of enzyme is 0.5 mgenzyme/g dry matter weight or lower, 0.4 mg enzyme composition/g drymatter weight or lower, 0.3 mg enzyme/g dry matter weight or lower, 0.25mg enzyme/g dry matter weight or lower, 0.20 mg enzyme/g dry matterweight or lower, 0.18 mg enzyme/g dry matter weight or lower, 0.15 mgenzyme/g dry matter weight or lower or 0.10 mg enzyme/g dry matterweight or lower (expressed as total of cellulase enzymes in mg enzyme/gdry matter). Low enzyme dosage is possible, since because of theactivity and stability of the enzymes, it is possible to increase thehydrolysis reaction time.

In a further aspect of the invention, the hydrolysis reaction time is 5hours or more, 10 hours or more, 20 hours or more, 40 hours or more, 50hours or more, 60 hours or more, 70 hours or more, 80 hours or more, 90hours or more, 100 hours or more, 120 hours or more, 130 h or more. Inanother aspect, the hydrolysis reaction time is 5 to 150 hours, 40 to130 hours, 50 to 120 hours, 60 to 120 hours, 60 to 110 hours, 60 to 100hours, 70 to 100 hours, 70 to 90 hours or 70 to 80 hours. Due to thestability of the enzyme composition longer hydrolysis reaction times arepossible with corresponding higher sugar yields.

The pH during hydrolysis may be chosen by the skilled person. In afurther aspect of the invention, the pH during the hydrolysis may be 3.0to 6.4. The stable enzymes of the invention may have a broad pH range ofup to 2 pH units, up to 3 pH units, up to 5 pH units. The optimum pH maylie within the limits of pH 2.0 to 8.0, 3.0 to 8.0, 3.5 to 7.0, 3.5 to6.0, 3.5 to 5.0, 3.5 to 4.5, 4.0 to 4.5 or is about 4.2.

In a further aspect of the invention the hydrolysis step is conducteduntil 70% or more, 80% or more, 85% or more, 90% or more, 92% or more,95% or more of available sugar in the lignocellulosic material isreleased.

Significantly, a process of the invention may be carried out using highlevels of dry matter (of the lignocellulosic material) in the hydrolysisreaction. Thus, the invention may be carried out with a dry mattercontent of about 5 wt % or higher, about 8 wt % or higher, about 10 wt %or higher, about 11 wt % or higher, about 12 wt % or higher, about 13 wt% or higher, about 14 wt % or higher, about 15 wt % or higher, about 20wt % or higher, about 25 wt % or higher, about 30 wt % or higher, about35 wt % or higher or about 40 wt % or higher. In a further embodiment,the dry matter content in the hydrolysis step is 14 wt %, 15 wt %, 16 wt%, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt%, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt%, 33 wt % or more or 14 to 33 wt %.

In another embodiment the dry matter content at the end of thehydrolysis is 5 wt % or higher, 6 wt % or higher, 7 wt % or higher, 8 wt% or higher, 9 wt % or higher, 10 wt % or higher, 11 wt % or higher, 12wt % or higher, 13 wt % or higher, 14 wt % or higher, 15 wt % or higher,16 wt % or higher, 17 wt % or higher, 18 wt % or higher, 19 wt % orhigher, 20 wt % or higher, 21 wt % or higher, 22 wt % or higher, 23 wt %or higher, 24 wt % or higher, 25 wt % or higher, 26 wt % or higher, 27wt % or higher, 28 wt % or higher, 29 wt % or higher, 30 wt % or higher,31 wt % or higher, 32 wt % or higher, 33 wt % or higher, 34 wt % orhigher, 35 wt % or higher, 36 wt % or higher, 37 wt % or higher, 38 wt %or higher or 39 wt % or higher. In another embodiment the dry mattercontent at the end of the enzymatic hydrolysis is between 5 wt %—40 wt%, 6 wt %—40 wt %, 7 wt %—40 wt %, 8 wt %—40 wt %, 9 wt %—40 wt %, 10 wt%—40 wt %, 11 wt %—40 wt %, 12 wt %—40 wt %, 13 wt %—40 wt %, 14 wt %—40wt %, 15 wt %—40 wt %, 16 wt %—40 wt %, 17 wt %—40 wt %, 18 wt %—40 wt%, 19 wt %—40 wt %, 20 wt %—40 wt %, 21 wt %—40 wt %, 22 wt %-40 wt %,23 wt %—40 wt %, 24 wt %—40 wt %, 25 wt %—40 wt %, 26 wt %—40 wt %, 27wt %-40 wt %, 28 wt %—40 wt %, 29 wt %—40 wt %, 30 wt %—40 wt %, 31 wt%—40 wt %, 32 wt %—40 wt %, 33 wt %—40 wt %, 34 wt %—40 wt %, 35 wt %—40wt %, 36 wt %—40 wt %, 37 wt %—40 wt %, 38 wt %—40 wt %, 39 wt %—40 wt%.

Fermentation

The process according to the invention may comprise a fermentation step.The fermentation can be done simultaneously with the hydrolysis in onereactor (SSF). Preferably the fermentation is done after the hydrolysisand optimal conditions for both hydrolysis and fermentation can beselected which might be different for hydrolysis and fermentation. In afurther aspect, the invention thus includes in step fermentationprocesses in which a microorganism is used for the fermentation of acarbon source comprising sugar(s), e.g. glucose, L-arabinose and/orxylose. The carbon source may include any carbohydrate oligo- or polymercomprising L-arabinose, xylose or glucose units, such as e.g.lignocellulose, xylans, cellulose, starch, arabinan and the like. Forrelease of xylose or glucose units from such carbohydrates, appropriatecarbohydrases (such as xylanases, glucanases, amylases and the like) maybe added to the fermentation medium or may be produced by the modifiedhost cell. In the latter case the modified host cell may be geneticallyengineered to produce and excrete such carbohydrases. An additionaladvantage of using oligo- or polymeric sources of glucose is that itenables to maintain a low(er) concentration of free glucose during thefermentation, e.g. by using rate-limiting amounts of the carbohydrases.This, in turn, will prevent repression of systems required formetabolism and transport of non-glucose sugars such as xylose. In apreferred process the modified host cell ferments both the L-arabinose(optionally xylose) and glucose, preferably simultaneously in which casepreferably a modified host cell is used which is insensitive to glucoserepression to prevent diauxic growth. In addition to a source ofL-arabinose, optionally xylose (and glucose) as carbon source, thefermentation medium will further comprise the appropriate ingredientrequired for growth of the modified host cell. Compositions offermentation media for growth of microorganisms such as yeasts orfilamentous fungi are well known in the art.

The fermentation time may be shorter than in conventional fermentationat the same conditions, wherein part of the enzymatic hydrolysis stillhas to take part during fermentation. In one embodiment, thefermentation time is 100 hours or less, 90 hours or less, 80 hours orless, 70 hours or less, or 60 hours or less, for a sugar composition of50 g/l glucose and corresponding other sugars from the lignocellulosicfeedstock (e.g. 50 g/l xylose, 35 g/l L-arabinose and 10 g/l galactose.For more dilute sugar compositions the fermentation time maycorrespondingly be reduced.

The fermentation process may be an aerobic or an anaerobic fermentationprocess. An anaerobic fermentation process is herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than 5, 2.5 or 1mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e. oxygenconsumption is not detectable), and wherein organic molecules serve asboth electron donor and electron acceptors. In the absence of oxygen,NADH produced in glycolysis and biomass formation, cannot be oxidised byoxidative phosphorylation. To solve this problem many microorganisms usepyruvate or one of its derivatives as an electron and hydrogen acceptorthereby regenerating NAD+. Thus, in a preferred anaerobic fermentationprocess pyruvate is used as an electron (and hydrogen acceptor) and isreduced to fermentation products such as ethanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol,ethylene, glycerol, butanol, a β-lactam antibiotics and a cephalosporin.In a preferred embodiment, the fermentation process is anaerobic. Ananaerobic process is advantageous since it is cheaper than aerobicprocesses: less special equipment is needed. Furthermore, anaerobicprocesses are expected to give a higher product yield than aerobicprocesses. Under aerobic conditions, usually the biomass yield is higherthan under anaerobic conditions. As a consequence, usually under aerobicconditions, the expected product yield is lower than under anaerobicconditions.

In another embodiment, the fermentation process is under oxygen-limitedconditions. More preferably, the fermentation process is aerobic andunder oxygen-limited conditions. An oxygen-limited fermentation processis a process in which the oxygen consumption is limited by the oxygentransfer from the gas to the liquid. The degree of oxygen limitation isdetermined by the amount and composition of the ingoing gas flow as wellas the actual mixing/mass transfer properties of the fermentationequipment used. Preferably, in a process under oxygen-limitedconditions, the rate of oxygen consumption is at least 5.5, morepreferably at least 6 and even more preferably at least 7 mmol/L/h.

The fermentation process is preferably run at a temperature that isoptimal for the modified cell. Thus, for most yeasts or fungal cells,the fermentation process is performed at a temperature which is lessthan 42° C., preferably less than 38° C. For yeast or filamentous fungalhost cells, the fermentation process is preferably performed at atemperature which is lower than 35, 33, 30 or 28° C. and at atemperature which is higher than 20, 22, or 25° C.

In an embodiment of the invention, in step the fermentation is conductedwith a microorganism that is able to ferment at least one C5 sugar. Inan embodiment the process is a process for the production of ethanolwhereby the process comprises the step comprises fermenting a mediumcontaining sugar(s) with a microorganism that is able to ferment atleast one C5 sugar, whereby the host cell is able to ferment glucose,L-arabinose and xylose to ethanol. The microorganism may be aprokaryotic or eukaryotic organism. The microorganism used in theprocess may be a genetically engineered microorganism. Examples ofsuitable organisms are yeasts, for instance Saccharomyces, e.g.Saccharomyces cerevisiae, Saccharomyces pastorianus or Saccharomycesuvarum, Hansenula, Issatchenkia, e.g. Issatchenkia orientalis, Pichia,e.g. Pichia stipites or Pichia pastoris, Kluyveromyces, e.g.Kluyveromyces fagilis, Candida, e.g. Candida pseudotropicalis or Candidaacidothermophilum, Pachysolen, e.g. Pachysolen tannophilus or bacteria,for instance Lactobacillus, e.g. Lactobacillus lactis, Geobacillus,Zymomonas, e.g. Zymomonas mobilis, Clostridium, e.g. Clostridiumphytofermentans, Escherichia, e.g. E. coli, Klebsiella, e.g. Klebsiellaoxytoca. In an embodiment thereof the microorganism that is able toferment at least one C5 sugar is a yeast. In an embodiment, the yeast isbelongs to the genus Saccharomyces, preferably of the speciesSaccharomyces cerevisiae, in which genetic modifications have been made.An example of such a microorganism and its preparation is described inmore detail in WO 2008/041840 and in European Patent ApplicationEP10160622.6, filed 21 Apr. 2010. In an embodiment, the fermentationprocess for the production of ethanol is anaerobic. Anaerobic hasalready been defined earlier herein. In another preferred embodiment,the fermentation process for the production of ethanol is aerobic. Inanother preferred embodiment, the fermentation process for theproduction of ethanol is under oxygen-limited conditions, morepreferably aerobic and under oxygen-limited conditions. Oxygen-limitedconditions have already been defined earlier herein.

In such process, the volumetric ethanol productivity is preferably atleast 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0 or 10.0 g ethanol per litre perhour. The ethanol yield on L-arabinose and optionally xylose and/orglucose in the process preferably is at least 20, 25, 30, 35, 40, 45,50, 60, 70, 80, 90, 95 or 98%. The ethanol yield is herein defined as apercentage of the theoretical maximum yield, which, for glucose andL-arabinose and optionally xylose is 0.51 g. ethanol per g. glucose orxylose.

In one aspect the fermentation process leading to the production ofethanol has several advantages by comparison to known ethanolfermentations processes:

-   -   anaerobic processes are possible;    -   oxygen limited conditions are also possible;    -   higher ethanol yields and ethanol production rates can be        obtained;    -   the strain used may be able to use L-arabinose and optionally        xylose.

Alternatively to the fermentation processes described above, at leasttwo distinct cells may be used, this means this process is aco-fermentation process. All preferred embodiments of the fermentationprocesses as described above are also preferred embodiments of thisco-fermentation process: identity of the fermentation product, identityof source of L-arabinose and source of xylose, conditions offermentation (aerobic or anaerobic conditions, oxygen-limitedconditions, temperature at which the process is being carried out,productivity of ethanol, yield of ethanol).

The fermentation process may be carried out without any requirement toadjust the pH during the process. That is to say, the process is onewhich may be carried out without the addition of any acid(s) or base(s).However, this excludes a pretreatment step, where acid may be added. Thepoint is that the composition of the invention is capable of acting atlow pH and, therefore, there is no need to adjust the pH of acid of anacid pretreated feedstock in order that saccharification or hydrolysismay take place. Accordingly, a method of the invention may be a zerowaste method using only organic products with no requirement forinorganic chemical input.

Overall Reaction Time

According to the invention, the overall reaction time (or the reactiontime of hydrolysis step and fermentation step together) may be reduced.In one embodiment, the overall reaction time is 300 hours or less, 200hours or less, 150 hours or less, 140 hours or less, 130 or less, 120hours or less, 110 hours or less, 100 hours of less, 90 hours or less,80 hours or less, 75 hours or less, or about 72 hours at 90% glucoseyield. Correspondingly lower overall times may be reached at lowerglucose yield.

Fermentation Products

Fermentation products which may be produced according to the inventioninclude amino acids, vitamins, pharmaceuticals, animal feed supplements,specialty chemicals, chemical feedstocks, plastics, solvents, fuels, orother organic polymers, lactic acid, and ethanol, including fuel ethanol(the term “ethanol” being understood to include ethyl alcohol ormixtures of ethyl alcohol and water).

Specific value-added products that may be produced by the methods of theinvention include, but not limited to, biofuels (including biogas,ethanol and butanol); lactic acid; 3-hydroxy-propionic acid; acrylicacid; acetic acid; 1,3-propane-diol; ethylene; glycerol; a plastic; aspecialty chemical; an organic acid, including citric acid, succinicacid and maleic acid; a solvent; an animal feed supplement; apharmaceutical such as a β-lactam antibiotic or a cephalosporin; avitamin; an amino acid, such as lysine, methionine, tryptophan,threonine, and aspartic acid; an enzyme, such as a protease, acellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, anoxidoreductase, a transferase or a xylanase; a chemical feedstock; or ananimal feed supplement.

Fermentation products that may be produced by the processes of theinvention can be any substance derived from fermentation, They include,but are not limited to, alcohols (such as arabinitol, butanol, ethanol,glycerol, methanol, 1,3-propanediol, sorbitol, and xylitol); organicacid (such as acetic acid, acetonic acid, adipic acid, ascorbic acid,acrylic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid,fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaricacid, 3-hydroxypropionic acid, itaconic acid, lactic acid, maleic acid,malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid,succinic acid, and xylonic acid); ketones (such as acetone); amino acids(such as aspartic acid, glutamic acid, glycine, lysine, serine,tryptophan, and threonine); alkanes (such as pentane, hexane, heptane,octane, nonane, decane, undecane, and dodecane), cycloalkanes (such ascyclopentane, cyclohexane, cycloheptane, and cyclooctane), alkenes (suchas pentene, hexene, heptene, and octene); and gases (such as methane,hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). Thefermentation product can also be a protein, a vitamin, a pharmaceutical,an animal feed supplement, a specialty chemical, a chemical feedstock, aplastic, a solvent, ethylene, an enzyme, such as a protease, acellulase, an amylase, a glucanase, a lactase, a lipase, a lyase, anoxidoreductase, a transferase or a xylanase.

Separation of Fermentation Product

The process according to the invention optionally comprises recovery offermentation product. A fermentation product may be separated from thefermentation broth in any known manner. For each fermentation productthe skilled person will thus be able to select a proper separationtechnique. For instance ethanol may be separated from a yeastfermentation broth by distillation, for instance steamdistillation/vacuum distillation in conventional way.

Certain embodiments of the invention will below be described in moredetail, but are in no way limiting the scope of the present invention.

Use of Thermostable Enzymes Under Optimal Temperature Conditions

In one embodiment, the invention relates to the use of thermostableenzymes such as cellulolytic enzymes of Rasamsonia for the production ofreducing sugars from pre-treated lignocellulosic feedstock in, but notlimiting to, ethanol production. Cellulolytic enzymes of Rasamsoniaapplied on pre-treated lignocellulosic feedstock showed maximalconversion rates at temperature within the range of 50 to 70° C. Theenzyme remains active under these circumstances for 14 days and morewithout complete cessation of activity.

By using optimal temperature conditions, maximal amount of reducingsugars can be released from feedstock (total hydrolysis) within theshortest possible hydrolysis time. In this way, 100% conversion ofcellulose in glucose is achieved in less than 5 days.

The theoretical maximum yield (Yps max in g product per gram glucose) ofa fermentation product can be derived from textbook biochemistry. Forethanol, 1 mole of glucose (180 g) yields according to normal glycolysisfermentation pathway in yeast 2 moles of ethanol (=2×46=92 g ethanol.The theoretical maximum yield of ethanol on glucose is therefore92/180=0.511 g ethanol/g glucose.

For butanol (MW 74 g/mole) or iso butanol, the theoretical maximum yieldis 1 mole of butanol per mole of glucose. So Yps max for(iso-)butanol=74/180=0.411 g (iso-)butanol/g glucose.

For lactic acid the fermentation yield for homolactic fermentation is 2moles of lactic acid (MW=90 g/mole) per mole of glucose. According tothis stoichiometry, the Yps max=1 g lactic acid/g glucose.

For other fermentation products a similar calculation may be made.

The cost reduction achieved with applying cellulolytic enzymes ofRasamsonia will be the result of an overall process time reduction.

Compensation of Lower Enzyme Dosage with Extended Hydrolysis Time UsingRasamsonia Enzymes

Due to the high stability of the stable enzymes, the activities do notcease in time, although less reducing sugars are liberated in the courseof the hydrolysis. It is possible to lower the enzyme dosage and extendthe use of the enzyme by prolonging the hydrolysis times to obtainsimilar levels of released reducing sugars. For example, 0.175 mLenzyme/g feedstock dry-matter resulted in release of approximately 90%of the theoretical maximum of reducing sugars from pre-treated feedstockwithin 72 h. When using 0.075 mL enzyme/g feedstock dry-matter,approximately 90% conversion of the theoretical maximum is achievedwithin 120 h. The results show that, because of the stability of theenzyme activity, lowering the enzyme dosage can be compensated byextending the hydrolysis time to obtain the same amount of reducingsugars. The same holds for hydrolysis of pre-treated feedstock atdry-matter contents higher than 10% shows that compensating effect ofextended hydrolysis time at 15% dry matter feedstock.

The cost reduction achieved by using stable cellulolytic enzymes, suchas of Rasamsonia, results from requiring less enzyme dosage, resultingin similar hydrolysis conversion yields.

Lowering the Risk on Contamination with Stable Enzymes

In a common process for converting lignocellulosic material intoethanol, process steps are preferably done under septic conditions tolower the operational costs. Contamination and growth of contaminatingmicroorganisms can therefore occur and result in undesirable sideeffects, such lactic acid, formic acid and acetic acid production, yieldlosses of ethanol on substrate, production of toxins and extracellularpolysaccharides, which may affect production costs significantly. A highprocess temperature and/or a short process time will limit the risk oncontamination during hydrolysis and fermentation. Thermostable enzymes,like those of Rasamsonia, are capable of hydrolysing lignocellulosicfeedstock at temperatures of higher than 60° C. At these temperatures,the risk that a contaminating microorganism will cause undesired sideeffects will be little to almost zero.

During the fermentation step, in which ethanol is produced, temperaturesare typically between 30 to 37° C. and will preferably not be raisedbecause of production losses. By applying fermentation process times asshort as possible the risks and effects of contamination and/or growthof contaminants will be reduced as much as possible. With stableenzymes, like those of Rasamsonia, a short as possible fermentationtimes can be applied (see description above), and thus risks oncontamination and/or growth of contaminants will be reduced as much aspossible. The cost reduction achieved with applying thermostablecellulolytic enzymes of Rasamsonia in this way will result from lowerrisk of process failures due to contamination.

Stable Enzymes Reduce Cooling Costs and Increase Productivity of EthanolPlants

The first step after thermal pretreatment will be to cool the pretreatedfeedstock to temperatures where the enzymes are optimal active. On largescale, this is typically done by adding (cooled) water, which will,besides decreasing the temperature, reduce the dry-matter content. Byusing thermos stable enzymes, like those of Rasamsonia, cost reductioncan be achieved by the fact that (i) less cooling of the pretreatedfeedstock is required since higher temperatures are allowed duringhydrolysis, and (ii) less water will be added, which will increase thedry-matter content during hydrolysis and fermentation and thus increasethe ethanol production capacity (amount produced per time unit pervolume) of an ethanol plant. Also, by using thermostable enzymesaccording to the invention, like those of Rasamsonia, cost reduction mayalso be achieved by using cooling water having higher temperature thatthe water that is used in a process with non-thermostable enzyme.

Enzyme Recycling after Hydrolysis with Stable Enzymes

At the end of the hydrolysis, enzyme activities appear to be low sincelittle reducing sugars are released once almost all cellulose isconverted. The amount of enzymatic activity present, however, hasdecreased only a little, assumingly mainly due to absorption of theenzymes to the substrate. By applying solid-liquid separation afterhydrolysis, such as centrifugation, filtration, sedicantation, etcetera,60% or more e.g. 70% of the enzyme activity in solution can be recoveredand re-used for hydrolysis of a new pre-treated lignocellulosicfeedstock during the next hydrolysis.

Moreover, after solid-liquid separation the enzyme in solution can beseparated from the solution containing reducing sugars and otherhydrolysis products from the enzymatic actions. This separation can bedone by, but not limiting to, (ultra and micro)filtration,centrifugation, sedicantation, sedimentation, with or without firstadsorption of the enzyme to a carrier of any kind.

For example, after hydrolysis of pre-treated feedstock with 0.175 mL/gfeedstock dry matter enzyme load for 20 h, 50% of the theoreticalmaximum amount of reducing sugars is liberated and after the samehydrolysis for 72 h, 90% of the theoretical maximum amount of reducingsugars is liberated. By centrifugation and ultrafiltration, 60-70% ofthe enzyme activity was recovered in the retentate, while the filtratecontained more than 80% of the liberated reducing sugars. By re-usingthe retentate, either as it is or after further purification and/orconcentration, enzyme dosage during the next hydrolysis step can bereduced with 60 to 70%. The cost reduction achieved by using stablecellulolytic enzymes, such as of Rasamsonia, in this way results fromrequiring less enzyme dosage.

Enzyme Recycling after Hydrolysis in Combination with Enzyme Productionand Yeast-Cell Recycling with Stable Enzymes

The process including enzyme recycling after hydrolysis, as describedabove, can be combined with recycling of the ethanol producingmicroorganism after fermentation and with the use of the reducing sugarscontaining filtrate as a substrate (purified and/or concentrated ordiluted) in enzyme-production fermentation and as substrate for thecultivation of the ethanol-producing microorganism.

Enzyme Recycling after Vacuum Distillation with Stable Enzymes

The thermo stability of enzymes, like those from Rasamsonia, causesremaining cellulolytic activity after hydrolysis, fermentation andvacuum distillation in the thin stillage. The total activity of theenzyme is reduced during the three successive process steps. The thinstillage obtained after vacuum distillation can thus be re-used as asource of enzyme for a newly startedhydrolysis-fermentation-distillation process cycle of pre-treated wheatstraw conversion into ethanol. The thin stillage can be used either inconcentrated or (un)diluted form and/or purified and with or withoutadditional enzyme supplementation.

Enzyme Recycling in Combination with Enzyme Supplementation after VacuumDistillation with Thermostable Enzymes

In an optimal process, an amount of enzyme is supplemented into the thinstillage, before its re-use in a new process cycle, equal to the amountof activity lost during the three successive process steps of theprevious process cycle. In this way over-dosage of enzyme is avoided andthus most efficient use of enzyme is obtained.

Moreover, by providing high enzyme dosage in the first process cycle,and supplementing enzyme equal to the amount of activity lost during thethree successive process steps in the following process cycles, highestpossible hydrolysis rates can be obtained in each process cycleresulting in short hydrolysis times of less than 48 h in combinationwith most efficient use of enzymes.

Use of Stable Enzymes in Mixed Systems

By applying mixing during hydrolysis, enzymes come more often in contactwith substrates, which results in a more efficient use of the catalyticactivity. This will result in a lower enzyme dosages and thus in lowercosts, unless the mixing has a negative effect on the enzymes. Stableenzymes, like the thermostable enzymes from Rasamsonia, are robust andcan resist circumstances of (locally) high shear and temperatures, whichis the case during intensive mixing of slurries. The use of it in mixedsystems is therefore beneficial and will lead to dosage and thus costsreduction.

The invention is further described by the following examples, whichshould not be construed as limiting the scope of the invention.

EXAMPLES Experimental Information Strains

Suitable Rasamsonia strains that can be used in the present examples toshow the effect and advantages of the invention are for example TEC-101,TEC-147, TEC-192, TEC-201 or TEC-210. The strains are described in WO2011/000949.

Preparation of Acid Pre-Treated Corn Stover Substrate

Dilute-acid pre-treated corn stover (aCS) was obtained as described inSchell, D. J., Applied Biochemistry and Biotechnology (2003), vol.105-108, pp 69-85. A pilot scale pretreatment reactor was used operatingat steady state conditions of 190° C., 1 min residence time and aneffective H₂SO₄ acid concentration of 1.45% (w/w) in the liquid phase.

Protein Measurement Assays 1. Total Protein TCA Biuret

The method was a combination of precipitation of protein using trichloroacetic acid (TCA) to remove disturbing substances and allowdetermination of the protein concentration with the colorimetric Biuretreaction. In the Biuret reaction, a copper (II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptidebonds in an alkaline solution. A violet color indicates the presence ofproteins. The intensity of the color, and hence the absorption at 546nm, is directly proportional to the protein concentration, according tothe Beer-Lambert law. The standardisation was performed using BSA(Bovine Serum Albumine) and the protein content was expressed in gprotein as BSA equivalent/L or mg protein as BSA equivalent/ml. Theprotein content was calculated using standard calculation protocolsknown in the art, by plotting the OD546 versus the concentration ofsamples with known concentration, followed by the calculation of theconcentration of the unknown samples using the equation generated fromthe calibration line.

2. Individual Proteins Using PAGE Sample Pretreatment SDS-PAGE

Based on the estimated protein concentration of the samples thefollowing samples preparation was performed. To 10 μl sample 40 μlMilliQ water and 50 μl TCA (20%) was added to dilute the sample fivetimes (˜1 mg/ml) and precipitate the proteins. After 1 hour on ice thesample was centrifuged (10 minutes, 14000 rpm). The pellet was washedwith 500 μl aceton and centrifuged (10 minutes, 14000 rpm). The pelletwas treated as described below.

SDS-PAGE

The pellet was dissolved in 65 μl of the MilliQ water, 25 μl NuPAGE™ LDSsample buffer (4×) Invitrogen and 10 μl NuPAGE™ Sample Reducing agent(10×) Invitrogen. Prior to the deanuarion step the sample was diluted 5times using a mix of MilliQ; NuPAGE™ LDS sample buffer and 10 μl NuPAGE™Sample Reducing in the ratio of 65:25:10. After mixing, the samples wereincubated in a thermo mixer for 10 minutes at 70° C. The samplesolutions were applied on a 4-12% Bis-Tris gel (NuPAGE™ BisTris,Invitrogen). A sample (10 μl) of marker M12 (Invitrogen) was alsoapplied on the gel. The gel was run at 200 V for 50 minutes, using theXCELL Surelock, with 600 ml 20× diluted SDS buffer in the outer bufferchamber and 200 ml 20× diluted SDS buffer, containing 0.5 ml ofantioxidant (NuPAGE™ Invitrogen) in the inner buffer chamber. Afterrunning, the gel was rinsed twice with demineralised water the gels werefixed with 50% methanol/7% acetic acid solution for one hour and stainedwith Sypro Ruby (50 ml per gel) overnight. An image was made using theTyphoon 9200 (610 BP 30, Green (532 nm), PMT 600V, 100 micron) afterwashing the gel with MilliQ water.

Quantitative Analysis of the Protein

Using the Typhoon scanner the ratio between protein bands within a lanewas determined using standard methods known in the art. The sample wasapplied in triplicate and the gray values were determined using theprogram Image quant. Values are expressed as relative % protein to thetotal protein, calculated using the gray value of the selected proteinband relative to the total gray value all the protein bands.

Glucan Conversion Calculation:

glucan conversion (%)=(glucose (g/l)×100%)/(glucan (fraction onDM)×dm(g/kg)×1.1)

Wherein:

glucose (g/l)=glucose concentration in supernatant after hydrolysis.glucan (fraction on dm)=glucan content of the substrate beforepretreatment.dm (g/kg)=dry matter of hydrolysis (f.i. 20% dm=200 g/kg).1.1=weight increase due to water incorporation during hydrolysis.Example calculation:

glucose=60 g/l

glucan fraction=0.40(is 40% on dry matter)

dm=200 g/kg

glucan conversion example=(60*100)/(0.4×200×1.1)=68% conversion

Correction for evaporation is made if necessary. The concentration inthe supernatant is subsequently converted to the concentration per kghydrolysate.

Measurement of Gluconic Acid in Biomass Hydrolysates by UPLC-MS/MS

The assay is based on separation of gluconic acid with an UPLC columnand detection by means of MS/MS (based on negative electrosprayionization). In order to exclude errors caused by ion suppression,evaporation and injection effects, a labelled internal standard, namely¹³C₆-gluconic acid, is used.

Chemicals and Reference Compounds

Water used for sample preparation and UPLC-MS/MS analysis was filteredby a Millipore 0.22 μm filter. HPLC-grade acetonitrile was obtained fromMerck (Amsterdam, the Netherlands). 0.1% (v/v) formic acid in water and0.1% formic acid in acetonitrile were obtained from Biosolve B.V.(Valkenswaard, the Netherlands). Gluconic acid reference compound wasobtained from Sigma (Zwijndrecht, the Netherlands). Isotopicallylabelled gluconic acid (¹³C₆, used as internal standard) was custom madeby Buchem B.V. (Apeldoorn, the Netherlands).

Internal Standard Solution

A stock solution was made by weighing 10 mg ¹³C₆-gluconic acid in a 10mL volumetric flask and dissolving in 10 mL water (˜1 mg/mL). From thisstock solution a working solution was prepared by pipetting 100 μL ofthe internal standard stock solution and adding 9.99 mL water (c˜10μg/mL).

Standard Solutions

A stock solution of gluconic acid was made by weighing 5 mg gluconicacid and adding 10 mL water. The stock solution was further diluted bypipetting 20 μL of the stock solution and adding 980 μL water (dilution1, c˜10 μg/mL). A further dilution was made by pipetting 100 μL ofdilution 1 and adding 900 μL water (dilution 2, c˜1 μg/mL). Acalibration curve was made in HPLC vials according to Table 1 below.

Sample Preparation

Biomass hydrolysates were defrosted, if necessary, and diluted ten timeswith water by diluting 150 μL of sample in a Eppendorf vial with 1350 μLwater followed by centrifugation at 13000 rcf for 15 minutes. Theresulting supernatant was diluted fifty times with water by pipetting 20μL of supernatant in a HPLC vial and adding 100 μL internal standardworking solution and 880 μL water.

UPLC-MS/MS

Gluconic acid was analyzed on a Waters UPLC iClass system consisting ofa Waters iClass Binary Solvent Manager and a Water iClass Sample ManagerFTN connected to a Waters Xevo TQD mass spectrometer (Waters, Milford,Mass., USA). Chromatographic separation was achieved with a WatersAcquity UPLC BEH C18 column (150×2.1 mm, 1.8 μm) using a gradientelution with A) 0.1% (v/v) formic acid in water and B) 0.1% formic acidin acetonitrile as mobile phases. The 7 min gradient started with 1minute at 99% A followed by a linear decrease to 90% A in 2 minutes,then washing with 20% A for 2 minutes and re-equilibrating with 99% Afor 2 minutes. The flow rate was kept at 0.35 mL/min, using an injectionvolume of 5 μl and the column temperature was set to 40° C.

The mass spectrometer was operated in the negative ionization mode. Dataacquisition and peak integration were performed with Masslynx 4.1software (Waters). Gluconic acid and ¹³C₆-gluconic acid detection wasperformed in multiple reaction monitoring mode (MRM). The generalsettings were as follows: the ESI capillary voltage was 2.0 kV,extractor voltage 3.0 V, cone voltage 30 V. The desolvation gas(nitrogen) flow was 800 L/h with the temperature set at 350° C., thecone gas (nitrogen) flow was 50 L/h, and the source temperature was 150°C. The following MRM settings were used: gluconic acid m/z 195.0→129.0,dwell time 0.1 s, collision voltage 2 V; ¹³C₆-gluconic acid m/z201.0→134.0, dwell time 0.08 s, collision voltage 2 V.

Quantification

The concentration of gluconic acid in g/L was calculated using linearregression:

${g\text{/}l\mspace{14mu} {compound}} = \frac{\begin{matrix}{\left( {\left( {{Area}\mspace{14mu} {{compound}/{Area}}\mspace{14mu} {internal}\mspace{14mu} {standard}} \right) - {intercept}} \right)*} \\{1000\mspace{14mu} \left( {{dilution}\mspace{14mu} {factor}} \right)}\end{matrix}}{{Slope}\mspace{14mu} {of}\mspace{14mu} {calibration}\mspace{14mu} {line}*1000}$

The calculated amount gluconic acid in g/l can be converted intogluconic acid in g/kg glucan in the lignocellulosic material by means ofthe following calculation.

When using acid pretreated corn stover at a concentration of 20% (w/w)dm, at the end of hydrolysis for 120 hours, at pH 4.5 and 62° C. thereis a pellet volume of 6% (due to insoluble) and a supernatant volume of94%. The supernatant has a density of 1.07 kg/l and a glucan percentageof 36%. So, acid pretreated corn stover at a concentration of 20% (w/w)dm has 72 g glucan/kg hydrolysate.

Starting with, for instance, 0.5 g/l gluconic acid this gives0.5/1.07=0.47 g gluconic acid/kg supernatant (1.07 is density ofliquid). This corresponds with 0.47*0.94=0.44 g gluconic acid/kghydrolysate (pellet factor is 6% due to insoluble). When using acidpretreated corn stover at a concentration of 20% (w/w) dm, thiscorresponds with 0.44/0.072=6.1 g gluconic acid/kg glucan.

Example 1 Use of Oxygen During Hydrolysis to Control the Amount ofGluconic Acid Formed

Optimal enzymatic hydrolysis of pretreated cornstover having 20% drymatter (containing 36% glucan on dry matter) at a reactor temperature of60° C. and pH of 4.5 is obtained by keeping the gluconic acidconcentration between 0.7 and 1.5 g/l in the supernatant of thehydrolysate by the addition of oxygen. This corresponds to 9.7-20.8 ggluconic acid produced per kg glucan during the hydrolysis of glucan.The hydrolysis is performed with 2.5 mg/g dry matter feedstock ofTEC-210 cellulase enzyme composition (or cocktail). TEC-210 is producedaccording to the inoculation and fermentation procedures described in WO2011/000949.

Example 2 Use of Oxygen During Hydrolysis to Control the Amount ofGluconic Acid Formed

The enzymatic hydrolysis was performed using acid pretreated cornstover(aCS) feedstock at a concentration of 20% (w/w) dry matter (dm). Thefeedstock solution was prepared by the dilution of concentratedfeedstock slurry with water. The pH was adjusted to pH 4.5 with a 25%(w/w) NH₄OH-solution. The enzymatic hydrolysis was performed at 1 kgscale using a 1.5 liter reactor. The pH was controlled at 4.5 and thetemperature was controlled at 62° C. The dissolved oxygen during theprocess was controlled by headspace gas recycling and additional freshair (containing 20-21% oxygen).

Prior to enzyme addition, headspace gas was recylced at a gas flow of 3I/hour using a peristaltic pump and a sparger. Due to the fact that thefeedstock consumes oxygen through a chemical reaction, the DO levelreached a level of 0% DO within one hour resulting in an anaerobicfeedstock and a headspace which was completely depleted from oxygen. Theresulting inert headspace gas (oxygen-free) was used throughout theentire hydrolysis as carrier gas for the introduced fresh air.

Next, the cellulase enzyme cocktail TEC210 was added to the feedstock ata dosage of 3.75 mg (TCA protein)/g dm. TEC-210 was produced accordingto the inoculation and fermentation procedures described in WO2011/000949. The total hydrolysis time was 120 hours.

Fresh air was introduced into the recycle loop of inert headspace gasduring the entire hydrolysis process at a fresh air flow of 0-3-6-12-24or 48 ml per kg reaction mixture per hour, respectively, startingdirectly after enzyme addition. The DO was measured constantly in allexperiments. Samples were drawn at the start and end of the experimentfor glucose analysis by HPLC.

A parallel experiment was conducted in a shake flask to determine themaximum level of glucan hydrolysis. This maximum hydrolysis wasdetermined by incubating the feedstock at a very high enzyme dosage (50mg (TCA protein)/g dm) at similar conditions (pH, temperature and dm) ina surplus of oxygen containing headspace air. DO measurement in eachexperiment constantly showed 0% DO. This can be explained by directconsumption of oxygen after its transfer from the oxygen containingrecycle gas stream into the liquid phase in the reactor.

The results are presented in Table 2. The glucose production increase iscalculated by subtracting the glucose concentration (in g/l) at thestart of the hydrolysis from the glucose concentration (in g/l) at theend of the hydrolysis for each condition (i.e. fresh air flow (ml/kg/h)0-3-6-12-24-48-96) and dividing the respective values with the valuefound when the fresh air flow (ml/kg/h) is 0 (this value was set to be100%).

The data clearly demonstrate that higher amounts of glucose are formedwhen the amount of formed gluconic acid at the end of the hydrolysis iskept above 3 g/kg glucan in the lignocellulosic material.

Example 3 The Effect of Gluconic Acid on Enzymatic Hydrolysis ofLignocellulosic Feedstock

Twenty grams of pretreated corn stover were incubated in a 50 ml Greinertest tube for 50 hours at a dry matter level of 6% w/w, pH 4.5 at 62° C.in the presence of 5 mg of the cellulase enzyme cocktail TEC210 per gramof dry matter and in the presence of 0, 2, 4, 6, or 8 g/l gluconic acidto determine the inhibiting effect of gluconic acid on the hydrolysis ofglucan in lignocellulosic feedstock. The experiment is performed atmaximum oxygen saturation.

Samples were taken after 50 hours of incubation and the samples werecentrifuged for further analysis in an Eppendorf centrifuge type 5810for 8 minutes at 4000 rpm. The glucose concentration was measured in thesupernatant of each sample by means of HPLC using a Bio-Rad HPX87Hcolumn. The results are shown in Table 3. The glucose release aspresented in Table 3 is a direct measure for the enzymatic hydrolysis(glucan hydrolysis). A gluconic acid level of 2 g/l in the supernatantcorresponds with 89 g/kg glucan in the lignocellulosic material. Thiscan be calculated as described above with a density of 1.02 kg/l, apellet factor of 2% and a glucan content of 21.6 g glucan/kghydrolysate.

The results clearly indicate that higher concentrations of gluconic acidhave a negative effect on glucan hydrolysis.

TABLE 1 Calibration curve μL Final μL μL internal concen- standardstandard standard tration solution solution working μL Standard (μg/mL)dilution 1 dilution 2 solution water Std 1 ~0.1 100 100 800 Std 2 ~0.2200 100 700 Std 3 ~0.5 50 100 850 Std 4 ~1 100 100 800 Std 5 ~2 200 100700 Std 6 ~5 500 100 400

TABLE 2 The effect of addition of oxygen in the enzymatic hydrolysis oflignocellulosic feedstock. Amount of oxygen (introduced Gluconic acidGlucose Fresh through fresh (in g/kg glucan production air flow air inmmol/kg in lignocellulosic increase (in ml/kg/h) glucan/hour)* material)(in %) 0 0 2.96 — 3 0.36 3.33 12.5 6 0.72 3.82 34 12 1.43 3.82 31 242.86 4.93 28 48 5.71 6.40 41 96 11.42 8.99 53 *Oxygen = 24.5 l/mol at25° C. and glucan content is 72 g/kg hydrolysate

TABLE 3 The effect of gluconic acid on enzymatic hydrolysis. Gluconicacid added Gluconic acid (in g/kg glucan Glucose release (in g/l) inlignocellulosic material) (in %) 0 0 100 2 89 90 4 178 82 6 267 79 8 35677

1. A process for preparation of a sugar product from lignocellulosicmaterial, comprising: a) optionally, pretreatment of the lignocellulosicmaterial, b) optionally, washing of the optionally pretreatedlignocellulosic material, c) enzymatic hydrolysis of the optionallywashed and/or optionally pretreated lignocellulosic material using anenzyme composition comprising at least two cellulases and whereby theenzyme composition at least comprises LPMO, and d) optionally, recoveryof a glucose-containing composition, wherein the amount of formedgluconic acid at the end of the enzymatic hydrolysis by the oxidation byLPMO of the lignocellulosic material containing cellulose and/orcello-oligosaccharides is kept between 3 to 80 g/kg glucan present inthe lignocellulosic material by adding a suitable amount of oxygen afterthe pretreatment and before and/or during the enzymatic hydrolysis tothe lignocellulosic material.
 2. A process for preparation of afermentation product from lignocellulosic material, comprising: a)optionally, pre-treatment of the lignocellulosic material, b)optionally, washing of the optionally pretreated lignocellulosicmaterial, c) enzymatic hydrolysis of the optionally washed and/oroptionally pretreated lignocellulosic material using an enzymecomposition comprising at least two cellulases and whereby the enzymecomposition at least comprises LPMO, and optionally purifying thehydrolysed lignocellulosic material, d) fermentation of the hydrolysedlignocellulosic material to produce a fermentation product, and e)optionally, recovery of a fermentation product, wherein the amount offormed gluconic acid at the end of the enzymatic hydrolysis by theoxidation by LPMO of the lignocellulosic material containing celluloseand/or cello-oligosaccharides is kept between 3 to 80 g/kg glucanpresent in the lignocellulosic material by adding a suitable amount ofoxygen after the pretreatment and before and/or during the enzymatichydrolysis to the lignocellulosic material.
 3. A process according toclaim 2, wherein the fermentation is conducted with a microorganism thatis able to ferment at least one C5 sugar.
 4. A process according toclaim 3, wherein the microorganism is of the species Saccharomycescerevisiae, in which genetic modifications have been made.
 5. A processaccording to claim 1, wherein during the enzymatic hydrolysis (c),oxygen is added to the lignocellulosic material.
 6. A process accordingto claim 1, wherein the oxygen is added in the form of bubbles.
 7. Aprocess according to claim 1, wherein the reactor for the enzymatichydrolysis has a volume of 1 m³ or more.
 8. A process according to claim1, wherein the enzymatic hydrolysis time is 5 to 150 hours.
 9. A processaccording to claim 1, wherein the enzyme composition used retainsactivity for 30 hours or more.
 10. A process according to claim 1,wherein the hydrolysis is conducted at a temperature of 45° C. or more,optionally 50° C. or more, or optionally at a temperature of 55° C. ormore.
 11. A process according to claim 1, wherein the enzyme compositionis derived from a fungus, optionally a microorganism of the genusRasamsonia or the enzyme composition comprises a fungal enzyme,optionally a Rasamsonia enzyme.
 12. A process according to claim 1,wherein the dry matter content in the hydrolysis (c) is 10 wt % or more,optionally is 14 wt % or more, or optionally is 14 to 33 wt %.
 13. Aprocess according to claim 1, in which the enzymatic hydrolysis takesplace in a batch, fed batch and/or continuous culture reactor.
 14. Aprocess according to claim 1, wherein the enzyme composition is in theform of a whole fermentation broth of a fungus.
 15. A process accordingto claim 1, in which oxygen is introduced as an oxygen-containing gas,optionally air.